Treponema pallidum fused antigen and assay for anti-treponema pallidum antibodies using the same fused antigen

ABSTRACT

A  Treponema pallidum  fused antigen in which at least two surface antigens of  Treponema pallidum  are fused and an assay for anti- Treponema pallidum  antibodies, using the above  Treponema pallidum  fused antigen.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to Treponema pallidum fused antigenand an assay for anti-Treponema pallidum antibodies, using the samefused antigen.

[0003] 2. Discussion of Background

[0004] Syphilis is an infectious disease caused by Treponema pallidum.Treponema pallidum belongs to the order Spirochaetales. It has beenestablished that several kinds of surface antigens exist on the cellsurface as antigens of Treponema pallidum (The Journal of Immunology,Vol. 129, p. 833-838, 1982; The Journal of Immunology, Vol. 129, p.1287-1291, 1982; Journal of Clinical Microbiology, Vol. 21, p. 82-87,1985; Journal of Clinical Microbiology, Vol. 30, p. 115-122, 1992).

[0005] When the body is infected with Treponema pallidum (hereinafterreferred to as Tp), anti-Tp antibodies are evoked and produced in theblood by those surface antigens, so that the diagnosis of syphilis ismade by inspecting the presence or absence of the anti-Tp antibodies inthe blood.

[0006] Generally immunoassays using the antigen-antibody reactionbetween the Tp antigens and the anti-Tp antibodies in the blood areutilized in order to detect the presence or absence of the anti-Tpantibodies in the blood of the patient.

[0007] For conducting the immunoassay for the Tp antibodies, a largeamount of the Tp antigens is required. However, a large amount of Tpcannot be cultivated in vitro, so that conventionally Tp is inoculatedinto the testes of the rabbit and the Tp antigen is obtained from thetestes of the rabbit infected with Tp and purified for use (Acta PatholMicrobiol Scand [B], Vol. 83, p. 157-160, 1975).

[0008] The above-mentioned rabbit testicular inoculation method,however, has the problems that the Tp antigen is contaminated withimpurities since Tp is cultivated in vivo in the rabbits, and thecultivated Tp varies due to individual differences of rabbits, and it isextremely difficult to obtain a large amount of Tp antigen withexcellent reproducibility.

[0009] Recently, because of the development of genetic engineering, thetechnology of artificially mass producing the Tp antigens has beendeveloped by cloning a gene which encodes the surface antigen of Tp(Science, Vol. 216; p. 522-523, 1982; Infection and Immunity, Vol. 36,p. 1238-1241, 1982; Infection and Immunity, Vol. 41, p. 709-721, 1983;Infection and Immunity, Vol. 42, p. 435-445, 1983; Infection andImmunity, Vol. 42, p. 187-196, 1983; Journal of Bacteriology, Vol. 162,p. 1227-1237, 1985; Infection and Immunity, Vol. 54, p. 500-506, 1986;Infection and Immunity, Vol. 56, p. 71-78, 1988; Infection and Immunity,Vol. 57, p. 2612-2623, 1989; Infection and Immunity, Vol. 57,p.3708-3714, 1989; Molecular Microbiology, Vol. 4, p. 1371-1379, 1990;Infection and Immunity, Vol. 58, p. 1697-1704, 1990; Infection andImmunity, Vol. 61, p. 1202-1210, 1993; Laid-Open Patent Application2-500403).

[0010] By using the technology of genetic engineering, the Tp antigencan be mass produced without using living animals. However, with respectto some kinds of Tp surface antigens, almost no product is expressed byusing only the gene which encodes the same antigen itself.

[0011] Therefore, in the above-mentioned case, a method of expression ofthe desired antigen has been proposed using a fused gene which isprepared by joining a gene such as thioredoxin derived from Escherichiacoli (hereinafter referred to as TRX) or a glutathione S-transferasederived from Schisstosoma japonicum (hereinafter referred to as GST) anda gene of the desired material as disclcosed in Japanese Laid-OpenPatent Application 5-507209 and Japanese Laid-Open Patent Application1-503441.

[0012] It has been discovered that a GST15kDa antigen which is a fusedgene of GST and a 15kDa antigen which is one surface antigen of Tp, anda GST17kDa antigen which is a fused gene of GST and 17kDa antigen whichis another surface antigen of Tp, exhibit high sensitivity when used forthe assay of the anti-Tp antibody as disclosed in Japanese Laid-OpenPatent Application 7-63365.

[0013] Since Escherichia coli and Schisstosoma japonicum can live in thehuman body and therefore there are many people who have a factor in theblood which reacts with TRX or GST. In such a case, even if the personis not infected with Tp, a positive reaction is exhibited with respectto infection with Tp. Thus, it is evident that this has a serious effecton the diagnosis of infection with Tp.

[0014] At present, syphilis is completely curable because of thedevelopment of antibiotics. Therefore, it is desired that syphilis becured based on a quick and accurate diagnosis of syphilis. In order toachieve this, there is a keen demand for an assay of Tp antigens andanti-Tp antibodies, with high sensitivity and specificity.

SUMMARY OF THE INVENTION

[0015] It is therefore a first object of the present invention toprovide Treponema pallidum (Tp) fused antigens in which at least twosurface antigens of Treponema pallidum are fused.

[0016] A second object of the present invention is to provide an assaymethod for anti-Treponema pallidum antibodies, using the above-mentionedTreponema pallidum fused antigen.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] A more complete appreciation of the invention and many of theattendant advantages thereof will be readily obtained as the samebecomes better understood by reference to the following detaileddescription when considered in connection with the accompanyingdrawings, wherein:

[0018]FIG. 1 is a detailed diagram of an expression vector for pW6A.

[0019]FIG. 2 is a diagram showing the procedure for preparing a genewhich encodes a 47K antigen.

[0020]FIG. 3 is a diagram showing the procedure for preparing a genewhich encodes a 15K antigen and a 17K antigen.

[0021]FIG. 4 is a detailed diagram of an expression vector for atwo-antigen fused antigen.

[0022]FIG. 5 is a detailed diagram of an expression vector for athree-antigen fused antigen.

[0023]FIG. 6 is a detailed diagram of an expression vector for anantigen in which 15K is repeatedly fused.

[0024]FIG. 7 are electrophoresis diagrams of purified fused antigens.

[0025]FIG. 8 is an electrophoresis diagram of a fused antigen in which15K is repeatedly fused.

[0026]FIG. 9 is a Western Blotting diagram of a fused antigen in whichan expressed 15K is repeatedly fused.

[0027]FIG. 10 is a diagram of an electrophoresis for quantitativemeasurement of a fused antigen in which 15K was repeatedly fused.

[0028]FIG. 11 is a graph showing a working curve for the quantitativemeasurement of a band on an electrophoresed gel prepared by use of BSA.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] The inventors of the present invention expressed a Tp fusedantigen which is fused with several classes of surface antigens by usinga gene which encodes the Tp fused antigen. As a result, it wasdiscovered that expression is significantly increased in comparison withthe amount of conventional expression. Furthermore, it was discoveredthat by using the Tp fused antigen as the antigen for the assay of theanti-Tp antibody, the anti-Tp antibody can be detected with highersensitivity than that of an assay system using surface antigensindividually. The present invention is based on such surprisingdiscoveries.

[0030] Surface antigens with various molecular weights are present onthe surface of the cells of Tp. As representative surface antigens,antigens with molecular weights of 15 kDa, 17 kDa, 42 kDa and 47 kDa areknown (The Journal of Immunology, Vol. 129, p. 833-838, 1982; TheJournal of Immunology, Vol. 129, p. 1287-1291, 1982; Journal of ClinicalMicrobiology, Vol. 21, p. 82-87, 1985; Journal of Clinical Microbiology,Vol. 30, p. 115-122, 1992). These antigens are designated TpN15, TpN17,TpN44.5(a) and TpN47 by S. J. Norris et al. (Microbiological Reviews,Vol. 57, P. 750-779). Hereinafter, a Tp surface antigen with a molecularweight of 47kDa may be referred to as 47K, a Tp surface antigen with amolecular weight of 17kDa may be referred to as 17K, and a Tp surfaceantigen with a molecular weight of 15kDa may be referred to as 15K.

[0031] The genes which encode these surface antigens are already clonedso that such antigens are produced by genetic engineering. Furthermore,the sequences of amino acids of those genes are also determined(Molecular Microbiology, Vol. 4, p.1371-1379, 1990; Infection andImmunity, Vol. 61, p. 1202-1210, 1993; Infection and Immunity, Vol. 10,p. 1568-1576 1992; Infection and Immunity, Vol. 57, p. 3708-3714, 1989).

[0032] The surface antigens of Tp to be used for the fused antigens ofthe present invention as used hereinafter means all the surface antigenswhich are present on the surface of the cells of Tp. For example,antigens such as 47K, 17K and 15K are such surface antigens.

[0033] Furthermore, the surface antigens for use in the presentinvention can be appropriately modified to such an extent that theantigenicity thereof is not impaired. For example, there is a signalpeptide of about 20 amino acids in the N terminal portion of the Tpsurface antigens when those antigens are translated in the cell.However, in a native antigen, such a signal peptide is eliminated. It isconsidered that this is because the signal peptide is cut by a signalpeptidase II after the DNA translation of the surface antigens(Microbial Pathogenesis, Vol. 7, p. 175-188, 1989; Infection andImmunity, Vol. 60, p. 1202-1210, 1993; Molecular Microbiology, Vol. 4,p. 1371-1379, 1990; Infection and Immunity, Vol. 60, p. 1568-1576,1993).

[0034] In the fused antigens of the present invention, the antigenswhich can be employed include both antigens which contain such signalpeptides at the N-terminuses thereof, and antigens which do not containsuch signal peptides at the N-terminus thereof.

[0035] For example, it is reported that there is a possibility that afurther 9 amino acids are joined to the C-terminus of 434 amino acids in47K (Infection and Immunity, Vol. 60, p1568-1576, 1992). In such a case,PCR is performed using the following primers. The sense primer andanti-sense primer are designed so as not to contain the coding sequencefor the signal peptide, but so as to contain the coding sequence for the9 amino acids joined to the C-terminus, respectively.

[0036] The Tp fused antigens of the present invention are antigens thatare expressed by fusing the genes of these surface antigens together.When fusing the genes of these Tp surface antigens, any combination ofthe Tp surface antigens can be employed and there is no restriction tothe combination thereof, since either different antigens or the sameantigens may be combined.

[0037] For expressing the fused antigens of the present invention,conventional genetic engineering technology can be employed. To be morespecific, a genomic DNA is extracted from the Tp which is cultivated,for instance, in the testes of rabbits, and used as a template.

[0038] PCR is performed to obtain the DNA fragment which encodes eachsurface antigen. Each fragment is amplified using primers which areprepared based on the known DNA sequence. The resultant DNA fragmentsare fused by recombinant PCR method (or so-called assemble PCR) or withDNA ligase.

[0039] The host such as Escherichia coli is transformed using a vectorinto which this DNA fragment is inserted, and such a bacterium ispropagated, for instance, by culturing, whereby the expression of thefused antigen is carried out. Thereafter, a desired fused antigen isobtained through purification such as destruction of the host, orelectrophoresis (Molecular Cloning A LABORATORY MANUAL SECOND EDITION1989).

[0040] DNA fragments of the Tp antigen to be fused can be inserted intothe vector using restriction endonuclease recognition sites such as NdeI, Sac I, EcoR I, Sal I, Xho I, BamH I, Kpn I, and Hindi III in themulti-cloning site.

[0041] The DNA fragment can be inserted into the vector eitherseparately or with such DNA fragments being directly joined in advance,for instance, by recombinant PCR method (or so-called assemble PCR).

[0042] When DNA fragments are inserted into the vector using theserestriction endonuclease recognition sites, a short amino acid may becaught between the inserted DNA fragments as a linker. This linker canbe removed by the following steps. The DNA fragments are fused byrecombinant PCR method (or so-called assemble PCR), and the resultantfused DNA fragment is inserted into the multi-cloning sites of theexpression vector. Alternatively, the fused antigen may be expressedwithout removing the linker.

[0043] Examples of the Treponema pallidum fused antigen of the presentinvention are a Treponema pallidum fused antigen in which at least twosurface antigens of Treponema pallidum are fused, a Treponema pallidumfused antigen in which at least three surface antigens of Treponemapallidum are fused, and a Treponema pallidum fused antigen in which twoto three surface antigens of Treponema pallidum are fused.

[0044] In the above-mentioned Treponema pallidum fused antigen of thepresent invention, at least two surface antigens, at least three surfaceantigens, or two to three surface antigens may have a molecular weightselected from 47kDa, 17kDa and 15kDa.

[0045] Specific examples of the Treponema pallidum fused antigens of thepresent invention are Treponema pallidum fused antigens in which surfaceantigens thereof are fused in the order of the surface antigen with amolecular weight of 47kDa, the surface antigen with a molecular weightof 17kDa, and the surface antigen with a molecular weight of 15kDa inview of the sequence from an N-terminus thereof to a C-terminus thereof,which may be referred to as 47-17-15 fused antigen; Treponema pallidumfused antigens in which surface antigens thereof are fused in the orderof the surface antigen with a molecular weight of 47kDa, the surfaceantigen with a molecular weight of 15kDa, and the surface antigen with amolecular weight of 17kDa in view of the sequence from an N-terminusthereof to a C-terminus thereof, which may be referred to as 47-15-17fused antigen; Treponema pallidum fused antigens in which surfaceantigens thereof are fused in the order of the surface antigen with amolecular weight of 15kDa, the surface antigen with a molecular weightof 17kDa, and the surface antigen with a molecular weight of 47kDa inview of the sequence from an N-terminus thereof to a C-terminus thereof,which may be referred to as 15-17-47 fused antigen; Treponema pallidumfused antigens in which surface antigens thereof are fused in the orderof the surface antigen with a molecular weight of 15kDa, the surfaceantigen with a molecular weight of 47kDa, and the surface antigen with amolecular weight of 17kDa in view of the sequence from an N-terminusthereof to a C-terminus thereof, which may be referred to as 15-47-17fused antigen; Treponema pallidum fused antigens in which surfaceantigens thereof are fused in the order of the surface antigen with amolecular weight of 17kDa, the surface antigen with a molecular weightof 15kDa, and the surface antigen with a molecular weight of 47kDa inview of the sequence from an N-terminus thereof to a C-terminus thereof,which may be referred to as 17-15-47 fused antigen; Treponema pallidumfused antigens in which surface antigens thereof are fused in the orderof the surface antigen with a molecular weight of 17kDa, the surfaceantigen with a molecular weight of 47kDa, and the surface antigen with amolecular weight of 15kDa in view of the sequence from an N-terminusthereof to a C-terminus thereof, which may be referred to as 17-47-15fused antigen; Treponema pallidum fused antigens in which surfaceantigens thereof are fused in the order of the surface antigen with amolecular weight of 47kDa, and the surface antigen with a molecularweight of 17kDa in view of the sequence from an N-terminus thereof to aC-terminus thereof, which may be referred to as 47-17 fused antigen;Treponema pallidum fused antigens in which surface antigens thereof arefused in the order of the surface antigen with a molecular weight of47kDa, and the surface antigen with a molecular weight of 15kDa in viewof the sequence from an N-terminus thereof to a C-terminus thereof,which may be referred to as 47-15 fused antigen; Treponema pallidumfused antigens in which surface antigens thereof are fused in the orderof the surface antigen with a molecular weight of 17kDa, and the surfaceantigen with a molecular weight of 47kDa in view of the sequence from anN-terminus thereof to a C-terminus thereof, which may be referred to as17-47 fused antigen; Treponema pallidum fused antigens in which surfaceantigens thereof are fused in the order of the surface antigen with amolecular weight of 17kDa, and the surface antigen with a molecularweight of 15kDa in view of the sequence from an N-terminus thereof to aC-terminus thereof, which may be referred to as 17-15 fused antigen;Treponema pallidum fused antigens in which surface antigens thereof arefused in the order of the surface antigen with a molecular weight of15kDa, and the surface antigen with a molecular weight of 47kDa in viewof the sequence from an N-terminus thereof to a C-terminus thereof,which may be referred to as 15-47 fused antigen; Treponema pallidumfused antigens in which surface antigens thereof are fused in the orderof the surface antigen with a molecular weight of 15kDa, and the surfaceantigen with a molecular weight of 17kDa in view of the sequence from anN-terminus thereof to a C-terminus thereof, which may be referred to as15-17 fused antigen; Treponema pallidum fused antigens in which surfaceantigens thereof are fused in the order of the surface antigen with amolecular weight of 15kDa, and the surface antigen with a molecularweight of 15kDa in view of the sequence from an N-terminus thereof to aC-terminus thereof, which may be referred to as 15-15 fused antigen;Treponema pallidum fused antigens in which surface antigens thereof arefused in the order of the surface antigen with a molecular weight of15kDa, the surface antigen with a molecular weight of 15kDa, and thesurface antigen with a molecular weight of 15kDa in view of the sequencefrom an N-terminus thereof to a C-terminus thereof, which may bereferred to as 15-15-15 fused antigen; and Treponema pallidum fusedantigens in which surface antigens thereof are fused in the order of thesurface antigen with a molecular weight of 15kDa, the surface antigenwith a molecular weight of 15kDa, the surface antigen with a molecularweight of 15kDa, and the surface antigen with a molecular weight of15kDa in view of the sequence from an N-terminus thereof to a C-terminusthereof, which may be referred to as 15-15-15-15 fused antigen.

[0046] Generally, in an assay system of anti-Tp antibodies, a mixture ofthe main antigens of Tp such as 47K, 17K and 15K are used, so that thenecessary antigens have to be separately purified.

[0047] In sharp contrast to this, when a fused antigen of the presentinvention is employed, the fused antigen contains a plurality ofantigens, so that the fused antigen can be purified as a single antigenand is easily available.

[0048] Furthermore, 17K and 15K antigens are rarely expressed by usingthe respective genes which encode such antigens. Therefore, theseantigens are expressed as fused proteins with a protein such as TRX orGST. However, a fused antigen expressed by a fused gene containing aheteroantigen other than Tp antigen may induce an unexpectednon-specific reaction which is not derived from Tp antigen.

[0049] In sharp contrast to this, the antigen of the present inventionis the fused antigen to which only Tp antigens are fused, and free of aheteroantigen other than Tp antigen, and therefore is an excellentantigen for specifically detecting the anti-Tp antibody.

[0050] It is known that in an antibody assay for the diagnosis of virusinfection, that a fused antigen may be employed to which a plurality ofkinds of antigens is fused (Hepatology, Vol.14, p.381-387, 1991).

[0051] In contrast to this, the fused antigen of the present inventionexhibits a much higher reactivity with the anti-Tp antibody than thereactivity in the case where each surface antigen is individuallyallowed to react with the anti-Tp antibody, so that there can beprovided an anti-Tp antibody assay method with unconventionally highassay sensitivity by use of said fused antigen in the assay system forthe anti-Tp antibody.

[0052] Furthermore, in the present invention, the main surface antigens,i.e., the 17K and 15K surface antigens, can be expressed in asurprisingly increased quantity when they are converted into fusedantigens in combination with the Tp antigens, even though 17K and 15Ksurface antigens are rarely expressed either by using the gene whichencodes a 17K surface agent alone or by using a 15K surface antigenalone.

[0053] The fused antigens of the present invention can be used in ananti-Tp antibody assay. The anti-Tp antibody assay means an assay inwhich the above-mentioned fused antigen is used as the antigen for theassay of the anti-Tp antibody. As long as the assay is based on animmune reaction between the anti-Tp antibody in the specimen and thefused antigen, the reaction may be carried by any method.

[0054] Various immunoassays are known to those skilled in the art, whichmay be classified, based on the reaction mode, into an agglutinationmethod, a nephelometry method, a sandwich method, and a competitionmethod; and based on the kinds of labeled materials, into enzyme-labeledimmunoassay, fluorescent labeled immunoassay, luminescent labeledimmunoassay and radioimmunoassay.

[0055] Of these immunoassay methods, particularly preferable immunoassaymethods are the agglutination method which does not require exclusiveequipment, ELISA method based on the enzyme-labeled immunoassay, whichis suitable for handling many specimens, and the luminescent labeledimmunoassay which is developed so as to have high sensitivity and ishighly automated.

[0056] When the fused antigen of the present invention is used for theassay of anti-Tp antibody, for example, in the case of the agglutinationmethod, the fused antigen of the present invention is bound to carrierssuch as latex particles, gelatin particles or magnetic particles, andnon-specific absorption sites thereof are blocked. The fusedantigen-bound carriers are allowed to react with a specimen to be testedfor a predetermined period of time, and the amount of the anti-Tpantibody in the specimen can be measured, using a detection index as theturbidity of the agglutination formed by the immune reaction or anagglutination image formed by the immune reaction.

[0057] In the ELISA method, the fused antigen of the present inventionis bound to the wells of a micro titer plate, and the non-specificabsorption sites thereof are blocked.

[0058] The specimen is then added to the wells of the fusedantigen-bound micro titer plate and allowed to react with the fusedantigen for a predetermined period of time. The wells are then washed,and an antihuman immuno-globulin labeled with an enzyme such asperoxidase is then allowed to react therewith. The wells are thenwashed, and an enzyme reaction is carried out with the addition of asubstrate corresponding to the labeling enzyme, whereby the enzymeactivity is measured. Thus, the amount of the anti-Tp antibody in thespecimen can be measured.

[0059] The above-mentioned agglutination method and ELISA method areknown in the field of this technology. However, the assay of the presentinvention is not necessarily limited to the above-mentioned methods.

[0060] Examples of the specimens for use in the assay of the presentinvention include humors and diluted humors, such as serum of human oranimals, to be tested for the diagnosis of Tp.

[0061] Other features of this invention will become apparent in thecourse of the following description of reference examples, and exemplaryembodiments which are given for illustration of the invention and arenot intended to be limiting thereof.

Reference Example 1 Preparation of Anti-47K Monoclonal Antibody,Anti-17K Monoclonal Antibody, and Anti-15K Monoclonal Antibody

[0062] As a surface antigen of Tp, 47K was expressed in Escherichia coliand purified.

[0063] Spleen cells obtained from a mouse which was immunized with theabove recombinant antigen, and mouse myeloma cells were subjected tocell fusion, whereby a hybridoma was prepared.

[0064] This hybridoma was cultured and allowed to react with theabove-mentioned recombinant antigen by ELISA using the above recombinantantigen and by Western blotting using the above native antigen 47K, sothat a hybridoma which is capable of reacting with both the recombinantantigen and the native antigen 47K was screened. The thus screenedhybridoma was cloned and a cell line thereof was established.

[0065] The thus established clone was injected into the abdominal cavityof the mouse, and the ascites was obtained therefrom and purified,whereby an anti-47K monoclonal antibody (hereinafter referred to as47KMab) was obtained.

[0066] GST17K in which GST was fused to the N-terminus of 17K wasexpressed in Escherichia coli and purified.

[0067] Spleen cells obtained from a mouse which was immunized with theabove recombinant antigen, and mouse myeloma cells were subjected tocell fusion, whereby a hybridoma was prepared.

[0068] This hybridoma was cultured and allowed to react with theabove-mentioned recombinant antigen by ELISA using the above recombinantantigen and by Western blotting using the above native antigen 17K, sothat a hybridoma which is capable of reacting with both the recombinantantigen and the native antigen 17K was screened. The thus screenedhybridoma was cloned and a cell line thereof was established.

[0069] The thus established clone was injected into the abdominal cavityof the mouse, and the ascites was obtained therefrom and purified,whereby an anti-17K monoclonal antibody (hereinafter referred to as17KMab) was obtained.

[0070] GST15K in which GST was fused to the N-terminus of 15K wasexpressed in Escherichia coli and purified.

[0071] Spleen cells obtained from a mouse which was immunized with theabove recombinant antigen, and mouse myeloma cells were subjected tocell fusion, whereby a hybridoma was prepared.

[0072] This hybridoma was cultured and allowed to react with theabove-mentioned recombinant antigen by ELISA using the above recombinantantigen and by Western blotting using the above native antigen 15K, sothat a hybridoma which is capable of reacting with both the recombinantantigen and the native antigen 15K was screened. The thus screenedhybridoma was cloned and a cell line thereof was established.

[0073] The thus established clone was injected into the abdominal cavityof the mouse, and the ascites was obtained therefrom and purified,whereby an anti-15K monoclonal antibody (hereinafter referred to as15KMab) was obtained.

Reference Example 2 Preparation of Expression Vector pW6A

[0074] The DNA sequence which encodes tac promoter and GST was removedfrom Vector pGEX2T made by Pharmacia Biotec Co., Ltd, and the DNAsequence from the T7 promotor to gene 10 to the multi-cloning site tothe T7 transcriptional-terminator from pGEMEX-1 made by Promega Co.,Ltd. was inserted into the above removed pGEX2T.

[0075] From the thus obtained vector, the DNA sequence of gene 10 wasremoved, and the lac operator synthesized on a DNA synthesiser(Trademark “Model 381A” made by Applied Biosystem Co., Ltd.) wasinserted immediately after the T7 promotor, and the multi-cloning sitewas partly modified. The thus prepared expression vector was named“pW6A”. FIG. 1 shows a detailed diagram of pW6A.

Reference Example 3 Preparation of Primers for Preparation of Vectorsfor Expressing Varieties of Fused Antigens

[0076] The following primers were synthesized on the DNA synthesiser(Trademark “Model 381A” made by Applied Biosystem Co., Ltd.) in order toprepare each expression vector for preparing each fused antigen in thefollowing Examples 1 to 16 and Reference Example 4:5′------------------------------------------------3′ Primer 1 (Sense)(SEQ ID NO:1) Nde I 47K-5′ terminus 18 bases TAGCC CATATGGGCTCGTCTCATCATGAG (29 bases) Primer 2 (Antisense)   (SEQ ID NO:2)15K-5′ terminus 17 bases 47K-3′ terminus 20 bases ATAGAACTAAATGAACAAGACACACGGGATAGGACAC (37 bases) Primer 3 (Sense) (SEQ ID NO:3) 15K-5′terminus 17 bases TGTTCATTTAGTTCTATCCC (20 bases) Primer 4 (Antisense)(SEQ ID NO:4) 17K-5′ terminus 15 bases 15K-3′ terminus 20 basesTGTGCACGAGACACA CCTGCTAATAATGGCTTCCT (35 bases) Primer 5 (Sense) (SEQ IDNO:5) 17K-5′ terminus 20 bases TGTGTCTCGTGCACAACCGT (20 bases) Primer 6(Antisense) (SEQ ID NO:6) BamH I 17K-3′ terminus 21 bases GATCC GGATCCCTA TTTCTTTGTTTTTTTGAGCAC Termination Codon (35 bases) Primer 7 (Sense)(SEQ ID NO:7) Nde I 15K-5′ terminus 18 bases TAGCC CATATGTGTTCATTTAGTTCTATC (29 bases) Primer 8 (Sense) (SEQ ID NO:8) Nde I17K-5′ terminus 18 bases TAGCC CATATG TGTGTCTCGTGCACAACC (29 bases)Primer 9 (Antisense) (SEQ ID NO:9) BamH I 15K-3′ terminus 17 bases GATCCGGATCC CTA CCTGCTAATAATGGCTT Termination Codon (31 bases) Primer 10(Antisense) (SEQ ID NO:10) BamH I 47K-3′ terminus 17 bases GATCC GGATCCCTA AGACACACGGGATAGGA Termination Codon (31 bases) Primer 11 (Sense)(SEQ ID NO:11) Sal I 15K-5′ terminus 18 bases CGAGGC GTCGACTGTTCATTTAGTTCTATC (30 bases) Primer 12 (Sense) (SEQ ID NO:12) Sal I47K-5′ terminus 18 bases GAAC GTCGAC TGTGGCTCGTCTCATCAT (28 bases)Primer 13 (Antisense) (SEQ ID NO:13) Xho I 47K-3′ terminus 18 bases CTTGCTCGAG AGACACACGGGATAGGAC (28 bases) Primer 14 (Sense) (SEQ ID NO:14)Sal I 17K-5′ terminus 18 bases AGTA GTCGAC TGTGTCTCGTGCACAACC (28 bases)Primer 15 (Antisense) (SEQ ID NO:15) Xho I 17K-3′ terminus 21 bases CAGACTCGAG TTTCTTTGTTTTTTTGAGCAC (31 bases) Primer 16 (Antisense) (SEQ IDNO:16) Sal I 17K-3′ terminus 21 bases CAGA GTCGAC TTTCTTTGTTTTTTTGAGCAC(31 bases) Primer 17 (Antisense) (SEQ ID NO:17) Sal I 15K-3′ terminus 18bases GCTA GTCGAC CCTGCTAATAATGGCTTC (28 bases) Primer 18 (Antisense)(SEQ ID NO:18) Sac I 47K-3′ terminus 20 bases CGTA GAGCTCAGACACACGGGATAGGACAC (30 bases) Primer 19 (Sense) (SEQ ID NO:19) Sac I17K-5′ terminus 20 bases TAGC GAGCTC TGTGTCTCGTGCACAACCGT (30 bases)Primer 20 (Antisense) (SEQ ID NO:20) BamH I Base for encoding 9 aminoacids GATCC GGATCC CTA AGACACACGGGATAGGACACCCCTCTT termination Codon47K-3′ terminus 18 bases CTGGGCCACTACCTTCGC (59 bases) Primer 21(Antisense) (SEQ ID NO:21) Sal I 47K-3′ terminus 18 bases CTCTT GTCGACAGACACACGGGATAGGAC (29 bases) Primer 22 (Antisense) (SEQ ID NO:22) BamHI 15K-3′ terminus 18 bases CCGG GGATCC CCTGCTAATAATGGCTTC (28 bases)Primer 23 (Sense) (SEQ ID NO:23) BamH I 15K-5′ terminus 18 bases CCGGGGATCC TGTTCATTTAGTTCTATC (28 bases) Primer 24 (Antisense) (SEQ IDNO:24) Kpn I 15K-3′ terminus 18 bases CCGG GGTACC CTA CCTGCTAATAATGGCTTCTermination Codon (31 bases) Primer 25 (Antisense) (SEQ ID NO:25) Kpn I15K-3′ terminus 18 bases CCGG GGTACC CCTGCTAATAATGGCTTC (28 bases)Primer 26 (Sense) (SEQ ID NO:26) Kpn I 15K-5′ terminus 18 bases CCGGGGTACC TGTTCATTTAGTTCTATC (28 bases) Primer 27 (Antisense) (SEQ IDNO:27) Hind III 15K-3′ terminus 15 bases CCGG AAGCTT CTA CCTGCTAATAATGGCTermination Codon (28 bases) Primer 28 (Antisense) (SEQ ID NO:28) EcoR I17K-3′ terminus 21 bases GACT GAATTC TTTCTTTGTTTTTTTGAGCAC (31 bases)Primer 29 (Sense) (SEQ ID NO:29) EcoR 15K-5′ terminus 21 bases GGTGGAATTC TGTTCATTTAGTTCTATCCCG (31 bases)

Reference Example 4 Preparation of Tp47K, 17K and 15K Genes

[0077]Treponema pallium (Nicols strain), which was subcultured in thetestes of rabbit, was purified by Percoll density-gradientcentrifugation (Sex. Transm. Dis., Vol. 11, p.275-286, 1984), and agenomic DNA was extracted by SDS-proteinase K/phenol.chloroform method.

[0078] By the polymerase chain reaction (PCR) method, the DNA fragmentwhich encodes 15K was amplified, using Primer 7 prepared in ReferenceExample 3 as a sense primer, Primer 9 prepared in Reference Example 3 asan antisense primer, and the above extracted genomic DNA as a template(Molecular Microbiology, Vol. 4, p.1371-1379, 1990).

[0079] A DNA fragment which encodes 17K was amplified, using Primer 8prepared in Reference Example 3 as a sense primer, and Primer 6 preparedin Reference Example 3 as an antisense primer (Infection and Immunity,Vol. 61, p.1202-1210, 1993). Furthermore, a DNA fragment which encodes47K was amplified, using Primer 1 prepared in Reference Example 3 as asense primer, and Primer 20 prepared in Reference Example 3 as anantisense primer (Infection and Immunity, Vol. 60, p.1568-1576, 1992).

[0080] In Infection and Immunity, Vol. 60, p.1568-1576, 1992, it isreported that it is possible that 9 amino acids are further bonded tothe C-terminus of 434 amion acids in 47K. Therefore, as shown in FIG. 2,at the amplification of the DNA fragement of 47K, a DNA fragment whichencodes 47K was prepared by adding a DNA sequence corresponding to the 9amino acids to the antisense primer to be used.

[0081]FIG. 3 shows a DNA fragment for encoding 15K and a DNA fragmentfor encoding 17K.

EXAMPLE 1 Preparation of Expression Vector for 15-17 Fused Antigen

[0082] (a) As a sense primer, Primer 7 prepared in Reference Example 3was selected, and as an antisense primer, Primer 4 prepared in Example 3was selected.

[0083] A mixture of each of the above-mentioned Primers 7 and 4 at afinal concentration of 1 μM, 0.1 ng of the 15K gene prepared inReference Example 4, and 2.5 units of Taq polymerase (made by TaKaRaCo., Ltd.) was subjected to the PCR under a heat application cycle of94° C. for 1 minute, 55° C. for 2 minutes and 72° C. for 3 minutes, withthis heat application cycle being repeated 30 times, whereby a DNAfragment 1 was prepared.

[0084] (b) As a sense primer, Primer 5 prepared in Reference Example 3was selected, and as an antisense primer, Primer 6 prepared in Example 3was selected.

[0085] A mixture of each of the above-mentioned Primers 5 and 6 at afinal concentration of 1 μM, 0.1 ng of the 17K gene prepared inReference Example 4, and 2.5 units of Taq polymerase (made by TaKaRaCo., Ltd.) was subjected to PCR under the same heat application cycle asin the step (a) in Example 1, with this heat application cycle beingrepeated 30 times, whereby a DNA fragment 2 was prepared.

[0086] (c) A mixture of 1 ng of the DNA fragment 1 prepared in the step(a) in Example 1, 1 ng of the DNA fragement 2, each of Primers 7 and 6prepared in Reference Example 3 at a final concentration of 1 μM, and2.5 units of the above-mentioned Taq polymerase (made by TaKaRa Co.,Ltd.) was subjected to the PCR under a heat application cycle of 94° C.for 1 minute, 55° C. for 2 minutes and 72° C. for 3 minutes, with thisheat application cycle being repeated 40 times, whereby a DNA fragmentfor encoding a 15-17 fused antigen was prepared.

[0087] (d) The pW6A prepared in Reference Example 3 was subjected todigestion at 37° C. for 1 hour with 5 units of Nde I (made by NewEngland Biolab Co., Ltd.) and 5 units of BamH I (made by New EnglandBiolab Co., Ltd.).

[0088] The 15-17 fused antigen gene prepared in the step (c) in Example1 was also subjected to digestion at 37° C. for 1 hour with 5 units ofNde I (made by New England Biolab Co., Ltd.) and 5 units of BamH I (madeby New England Biolab Co., Ltd.).

[0089] (e) The pW6A and 15-17 fused antigen gene, which were subjectedto digestion in the step (d) in Example 1, were ligated at 16° C. for 2hours by use of Ligation Kit ver. 1 (made by TaKaRa Co., Ltd.). Afterthe completion of this reaction, Escherichia coli DH5α, which is acompetent cell prepared by Hanahan method, was transformed by use of theabove reaction mixture. The thus transformed Escherichia coli cells werethen plated on an LB agar plate containing 50 μg/ml ampicillin andincubated at 37° C. overnight. From the colonies of theampicillin-resistant Escherichia coli cells thus produced, thetransformant was selected carrying an expression vector for a 15-17fused antigen with which a 15-17 fused antigen gene was integrated. Thethus selected transformant was then cultured in an ampicillin-containingLB liquid culture medium, with shaking at 37° C. overnight. The desiredtransformant clone was harvested, from which a desired vector waspurified by an alkali SDS method.

[0090] The results are shown in FIG. 4.

EXAMPLE 2 Preparation of Expression Vector for 15-47 Fused Antigen

[0091] (a) As a sense primer, Primer 7 prepared in Reference Example 3was selected, and as an antisense primer, Primer 17 prepared in Example3 was selected.

[0092] A mixture of each of the above-mentioned Primers 7 and 17 at afinal concentration of 1 μM, 0.1 ng of the 15K gene prepared inReference Example 4, and 2.5 units of Taq polymerase (made by TaKaRaCo., Ltd.) was subjected to PCR under a heat application cycle of 94° C.for 1 minute, 55° C. for 2 minutes and 72° C. for 3 minutes, with thisheat application cycle being repeated 30 times, whereby a DNA fragment 3was prepared.

[0093] (b) The DNA fragment 3 prepared in the step (a) of Example 2 wassubjected to digestion at 37° C. for 1 hour with 5 units of Nde I (madeby New England Biolab Co., Ltd.) and 5 units of Sal I (made by ToyoboCo., Ltd.).

[0094] The pW6A prepared in Reference Example 2 was also subjected todigestion at 37° C. for 1 hour with 5 units of Nde I (made by NewEngland Biolab Co., Ltd.) and 5 units of Sal I (made by Toyobo Co.,Ltd.).

[0095] After these reactions, the DNA fragment 3 was inserted into thepW6A prepared in Reference Example 2 under the same conditions as in thestep (e) in Example 1.

[0096] (c) As a sense primer, Primer 12 prepared in Reference Example 3was selected, and as an antisense primer, Primer 10 prepared in Example3 was selected.

[0097] A mxture of each of the above-mentioned Primers 12 and 10 at afinal concentration of 1 μM, 0.1 ng of the 47K gene prepared inReference Example 4, and 2.5 units of the above-mentioned Taq polymerase(made by TaKaRa Co., Ltd.) was subjected to the PCR under a heatapplication cycle of 94° C. for 1 minute, 55° C. for 2 minutes and 72°C. for 3 minutes, with this heat application cycle being repeated 30times, whereby a DNA fragment 4 was prepared.

[0098] (d) The DNA fragment 3 inserted in pW6A prepared in the step (b)of Example 2 was subjected to digestion at 37° C. for 1 hour with 5units of the above-mentioned Sal I and 5 units of the above-mentionedBamH I.

[0099] The DNA fragment 4 prepared in the step (c) of Example 2 was alsosubjected to digestion at 37° C. for 1 hour with 5 units of theabove-mentioned Sal I and 5 units of the above-mentioned BamH I.

[0100] After these reactions, the DNA fragment 4 was inserted into theDNA fragment 3 inserted pW6A under the same conditions as in the step(e) in Example 1, whereby an expression vector for the 15-47 fusedantigen was obtained.

[0101] The results are shown in FIG. 4.

EXAMPLE 3 Preparation of Expression Vector for 17-47 Fused Antigen

[0102] (a) As a sense primer, Primer 8 prepared in Reference Example 3was selected, and as an antisense primer, Primer 15 prepared in Example3 was selected.

[0103] A mixture of each of the above-mentioned Primers 8 and 15 at afinal concentration of 1 μM, 0.1 ng of the 17K gene prepared inReference Example 4, and 2.5 units of the above-mentioned Taq polymerasewas subjected to PCR under a heat application cycle of 94° C. for 1minute, 55° C. for 2 minutes and 72° C. for 3 minutes, with this heatapplication cycle being repeated 30 times, whereby a DNA fragment 5 wasprepared.

[0104] (b) The DNA fragment 5 prepared in the step (a) of Example 3 wassubjected to digestion at 37° C. for 1 hour with 5 units of theabove-mentioned Nde I and 5 units of Xho I (made by Toyobo Co., Ltd.).

[0105] The pW6A prepared in Reference Example 2 was also subjected todigestion at 37° C. for 1 hour with 5 units of the above-mentioned Nde Iand 5 units of the above-mentioned Xho I.

[0106] After these reactions, the DNA fragment 5 was inserted into thepW6A under the same conditions as in the step (e) in Example 1.

[0107] (c) The DNA fragment 4 obtained in the step (c) of Example 2 wassubjected to digestion at 37° C. for 1 hour with 5 units of theabove-mentioned Sal I and 5 units of the above-mentioned BamH I.

[0108] The DNA fragment 5 inserted in pW6A obtained in the step (b) ofExample 3 was subjected to digestion at 37° C. for 1 hour with 5 unitsof the above-mentioned Xho I and 5 units of the above-mentioned BamH I.

[0109] After these reactions, the DNA fragment 4 was inserted into theDNA fragment 5 inserted pW6A under the same conditions as in the step(e) in Example 1, whereby an expression vector for the 17-47 fusedantigen was prepared.

[0110] The results are shown in FIG. 4.

EXAMPLE 4 Preparation of Expression Vector for 17-15 Fused Antigen

[0111] (a) As a sense primer, Primer 11 prepared in Reference Example 3was selected, and as an antisense primer, Primer 9 prepared in Example 3was selected.

[0112] A mixture of each of the above-mentioned Primers 11 and 9 at afinal concentration of 1 μM, 0.1 ng of the 15K gene prepared inReference Example 4, and 2.5 units of the above-mentioned Taq polymerasewas subjected to the PCR under a heat application cycle of 94° C. for 1minute, 55° C. for 2 minutes and 72° C. for 3 minutes, with this heatapplication cycle being repeated 30 times, whereby a DNA fragment 6 wasprepared.

[0113] (b) The DNA fragment 6 prepared in the step (a) of Example 4 wassubjected to digestion at 37° C. for 1 hour with 5 units of theabove-mentioned Sal I and 5 units of the above-mentioned BamH I.

[0114] The DNA fragment 5 inserted in pW6A prepared in the step (b) ofExample 3 was subjected to digestion at 37° C. for 1 hour with 5 unitsof the above-mentioned Xho I and 5 units of the above-mentioned BamH I.

[0115] After these reactions, the DNA fragment 6 was inserted into theDNA fragment 5 inserted pW6A under the same conditions as in the step(e) in Example 1, whereby an expression vector for the 17-15 fusedantigen was prepared.

[0116] The results are shown in FIG. 4.

EXAMPLE 5 Preparation of Expression Vector for 47-15 Fused Antigen

[0117] (a) As a sense primer, Primer 1 prepared in Reference Example 3was selected, and as an antisense primer, Primer 21 prepared in Example3 was selected.

[0118] A mixture of each of the above-mentioned Primers 1 and 21 at afinal concentration of 1 μM, 0.1 ng of the 47K gene prepared inReference Example 4, and 2.5 units of the above-mentioned Taq polymerasewas was subjected to the PCR under a heat application cycle of 94° C.for 1 minute, 55° C. for 2 minutes and 72° C. for 3 minutes, with thisheat application cycle being repeated 30 times, whereby a DNA fragment 7was prepared.

[0119] (b) The DNA fragment 7 prepared in the step (a) of Example 5 wassubjected to digestion at 37° C. for 1 hour with 5 units of theabove-mentioned Nde I and 5 units of the above-mentioned Sal I.

[0120] The pW6A prepared in Reference Example 2 was also subjected todigestion at 37° C. for 1 hour with 5 units of the above-mentioned Nde Iand 5 units of the above-mentioned Sal I.

[0121] After these reactions, the DNA fragment 7 was inserted into thepW6A under the same conditions as in the step (e) in Example 1.

[0122] (c) The DNA fragment 6 prepared in the step (a) of Example 4 wassubjected to digestion at 37° C. for 1 hour with 5 units of theabove-mentioned Sal I and 5 units of the above-mentioned BamH I.

[0123] The DNA fragment 7 inserted in pW6A prepared in the step (b) ofExample 5 was also subjected to digestion at 37° C. for 1 hour with 5units of the above-mentioned Sal I and 5 units of the above-mentionedBamH I.

[0124] After these reactions, the DNA fragment 6 was inserted into theDNA fragment 7 inserted in pW6A under the same conditions as in the step(e) in Example 1, whereby an expression vector for the 47-15 fusedantigen was prepared.

[0125] The results are shown in FIG. 4.

EXAMPLE 6 Preparation of Expression Vector for 47-17 Fused Antigen

[0126] (a) As a sense primer, Primer 14 prepared in Reference Example 3was selected, and as an antisense primer, Primer 6 prepared in Example 3was selected.

[0127] A mixture of each of the above-mentioned Primers 14 and 6 at afinal concentration of 1 μM, 0.1 ng of the 17K gene prepared inReference Example 4, and 2.5 units of the above-mentioned Taq polymerasewas subjected to the PCR under a heat application cycle of 94° C. for 1minute, 55° C. for 2 minutes and 72° C. for 3 minutes, with this heatapplication cycle being repeated 30 times, whereby a DNA fragment 8 wasprepared.

[0128] (b) The DNA fragment 8 prepared in the step (a) of Example 6 wassubjected to digestion at 37° C. for 1 hour with 5 units of theabove-mentioned Sal I and 5 units of the above-mentioned BamH I.

[0129] The DNA fragment 7 inserted pW6A prepared in the step (b) ofExample 5 was subjected to digestion at 37° C. for 1 hour with 5 unitsof the above-mentioned Sal I and 5 units of the above-mentioned BamH I.

[0130] After these reactions, the DNA fragment 8 was inserted into theDNA fragment 7 inserted pW6A under the same conditions as in the step(e) in Example 1, whereby an expression vector for the 47-17 fusedantigen was prepared.

[0131] The results are shown in FIG. 4.

EXAMPLE 7 Preparation of Expression Vector for 47-15-17 Fused Antigen

[0132] (a) As a sense primer, Primer 11 prepared in Reference Example 3was selected, and as an antisense primer, Primer 6 prepared in Example 3was selected.

[0133] A mixture of each of the above-mentioned Primers 11 and 6 at afinal concentration of 1 μM, 0.1 ng of the expression vector for the15-17 fused antigen prepared in Example 1, and 2.5 units of theabove-mentioned Taq polymerase was subjected to PCR under a heatapplication cycle of 94° C. for 1 minute, 55° C. for 2 minutes and 72°C. for 3 minutes, with this heat application cycle being repeated 30times, whereby a DNA fragment 9 was prepared.

[0134] (b) The DNA fragment 8 prepared in the step (a) of Example 7 wassubjected to digestion at 37° C. for 1 hour with 5 units of theabove-mentioned Sal I and 5 units of the above-mentioned BamH I.

[0135] The DNA fragment 7 inserted in pW6A prepared in the step (b) ofExample 5 was also subjected to digestion at 37° C. for 1 hour with 5units of the above-mentioned Sal I and 5 units of the above-mentionedBamH I.

[0136] After these reactions, the DNA fragment 9 was inserted into theDNA fragment 7 inserted pW6A under the same conditions as in the step(e) in Example 1, whereby an expression vector for 47-15-17 fusedantigen was prepared.

[0137] The results are shown in FIG. 5.

EXAMPLE 8 Preparation of Expression Vector for 17-47-15 Fused Antigen

[0138] (a) As a sense primer, Primer 12 prepared in Reference Example 3was selected, and as an antisense primer, Primer 2 prepared in Example 3was selected.

[0139] A mixture of each of the above-mentioned Primers 7 and 17 at afinal concentration of 1 μM, 0.1 ng of the 47K gene prepared inReference Example 4, and 2.5 units of the above-mentioned Taq polymerasewas subjected to

[0140] PCR under a heat application cycle of 94° C. for 1 minute, 55° C.for 2 minutes and 72° C. for 3 minutes, with this heat application cyclebeing repeated 30 times, whereby a DNA fragment 10 was prepared.

[0141] (b) As a sense primer, Primer 3 prepared in Reference Example 3was selected, and as an antisense primer, Primer 9 prepared in Example 3was selected.

[0142] A mixture of each of the above-mentioned Primers 3 and 9 at afinal concentration of 1 μM, 0.1 ng of the 15K gene prepared inReference Example 4, and 2.5 units of the above-mentioned Taq polymerasewas subjected to the PCR under a heat application cycle of 94° C. for 1minute, 55° C. for 2 minutes and 72° C. for 3 minutes, with this heatapplication cycle being repeated 30 times, whereby a DNA fragment 11 wasprepared.

[0143] (c) A mixture of 1 ng of the DNA fragment 10 prepared in the step(a) of Example 8, 1 ng of the DNA fragment 11 prepared in the step (b)of Example 8, each of the Primers 12 and 9 at a final concentration of 1μM, and 2.5 units of the above-mentioned Taq polymerase was subjected toPCR under a heat application cycle of 94° C. for 1 minute, 55° C. for 2minutes and 72° C. for 3 minutes, with this heat application cycle beingrepeated 30 times, whereby a gene for encoding the 47-15 fused gene wasprepared.

[0144] (d) The gene encoding the 47-15 fused gene prepared in the step(c) of Example 8 was subjected to digestion at 37° C. for 1 hour with 5units of the above-mentioned Sal I and 5 units of the above-mentionedBamH.

[0145] The DNA fragment 5 inserted pW6A prepared in the step (b) ofExample 3 was also subjected to digestion at 37° C. for 1 hour with 5units of the above-mentioned Xho I and 5 units of the above-mentionedBamH I.

[0146] After these reactions, a gene for encoding the 47-15 fusedantigen was inserted into the DNA fragment 5 inserted pW6A under thesame conditions as in the step (e) in Example 1, whereby an expressionvector for the 17-47-15 fused antigen was obtained.

[0147] The results are shown in FIG. 5.

EXAMPLE 9 Preparation of Expression Vector for 17-15-47 Fused Antigen

[0148] (a) As a sense primer, Primer 8 prepared in Reference Example 3was selected, and as an antisense primer, Primer 17 prepared in Example3 was selected.

[0149] A mixture of each of the above-mentioned Primers 8 and 17 at afinal concentration of 1 μM, 0.1 ng of the expression vector for the17-15 fused antigen prepared in Example 4, and 2.5 units of theabove-mentioned Taq polymerase was subjected to PCR under a heatapplication cycle of 94° C. for 1 minute, 55° C. for 2 minutes and 72°C. for 3 minutes, with this heat application cycle being repeated 30times, whereby a DNA fragment 12 was prepared.

[0150] (b) The DNA fragment 12 prepared in the step (a) of Example 9 wassubjected to digestion at 37° C. for 1 hour with 5 units of theabove-mentioned Nde I and 5 units of the above-mentioned Sal I.

[0151] The pW6A prepared in Reference Example 2 was also subjected todigestion at 37° C. for 1 hour with 5 units of the above-mentioned Nde Iand 5 units of the above-mentioned Sal I.

[0152] After these reactions, the DNA fragment 12 was inserted into pW6Aunder the same conditions as in the step (e) in Example 1.

[0153] (c) The DNA fragment 4 prepared in the step (c) of Example 2 wassubjected to digestion at 37° C. for 1 hour with 5 units of theabove-mentioned Sal I and 5 units of the above-mentioned BamH I.

[0154] The DNA fragment 12 inserted in pW6A prepared in the step (b) ofExample 9 was also subjected to digestion at 37° C. for 1 hour with 5units of the above-mentioned Sal I and 5 units of the above-mentionedBamH I.

[0155] After these reactions, the DNA fragment 4 was inserted into theDNA fragment 12 inserted pW6A under the same conditions as in the step(e) in Example 1, whereby an expression vector for a 17-15-47 fusedantigen was prepared.

[0156] The results are shown in FIG. 5.

EXAMPLE 10 Preparation of Expression vector for 15-47-17 Fused Antigen

[0157] (a) As a sense primer, Primer 12 prepared in Reference Example 3was selected, and as an antisense primer, Primer 13 prepared in Example3 was selected.

[0158] A mixture of each of ehe above-mentioned Primers 12 and 13 at afinal concentration of 1 μM, 0.1 ng of the 47K gene prepared inReference Example 4, and 2.5 units of the above-mentioned Taq polymerasewas subjected to PCR under a heat application cycle of 94° C. for 1minute, 55° C. for 2 minutes and 72° C. for 3 minutes, with this heatapplication cycle being repeated 30 times, whereby a DNA fragment 13 wasprepared.

[0159] (b) The DNA fragment 13 prepared in the step (a) of Example 10was subjected to digestion at 37° C. for 1 hour with 5 units of theabove-mentioned Sal I and 5 units of the above-mentioned Xho I.

[0160] The pW6A prepared in Reference Example 2 was also subjected todigestion at 37° C. for 1 hour with 5 units of the above-mentioned Sal Iand 5 units of the above-mentioned Xho I.

[0161] After these reactions, the DNA fragment 13 was inserted into thepW6A under the same conditions as in the step (e) in Example 1.

[0162] (c) The DNA fragment 8 prepared in Example 6 was subjected todigestion at 37° C. for 1 hour with 5 units of the above-mentioned Sal Iand 5 units of the above-mentioned BamH.

[0163] The DNA fragment 13 inserted pW6A prepared in the step (b) ofExample 10 was also subjected to digestion at 37° C. for 1 hour with 5units of the above-mentioned Xho I and 5 units of the above-mentionedBamH I.

[0164] After these reactions, the DNA fraction 8 was inserted into theDNA fragment 13 inserted pW6A under the same conditions as in the step(e) in Example 1, whereby a DNA fragment 13-8 inserted pW6A wasprepared.

[0165] (d) The DNA fragment 3 prepared in the step (a) of Example 2 wassubjected to digestion at 37° C. for 1 hour with 5 units of theabove-mentioned Nde I and 5 units of the above-mentioned Sal I.

[0166] The DNA fragment 13-8 inserted pW6A prepared in the step (c) ofExample 10 was also subjected to digestion at 37° C. for 1 hour with 5units of the above-mentioned Nde I and 5 units of the above-mentionedSal I.

[0167] After these reactions, the DNA fragment 3 was inserted into theDNA fragment 13-8 inserted pW6A under the same conditions as in the step(e) in Example 1, whereby an expression vector for a 15-47-17 fusedantigen was prepared.

EXAMPLE 11 Preparation of Expression Vector for 15-17-47 Fused Antigen

[0168] (a) As a sense primer, Primer 7 prepared in Reference Example 3was selected, and as an antisense primer, Primer 16 prepared in Example3 was selected.

[0169] A mixture of each of the above-mentioned Primers 7 and 16 at afinal concentration of 1 μM, 0.1 ng of the expression vector for the15-17 fused antigen prepared in Example 1, and 2.5 units of theabove-mentioned Taq polymerase was subjected to PCR under a heatapplication cycle of 94° C. for 1 minute, 55° C. for 2 minutes and 72°C. for 3 minutes, with this heat application cycle being repeated 30times, whereby a DNA fragment 14 was prepared.

[0170] (b) The DNA fragment 14 prepared in the step (a) of Example 11was subjected to digestion at 37° C. for 1 hour with 5 units of theabove-mentioned Nde I and 5 units of the above-mentioned Sal I.

[0171] The pW6A prepared in Reference Example 2 was also subjected todigestion at 37° C. for 1 hour with 5 units of the above-mentioned Nde Iand 5 units of the above-mentioned Sal I.

[0172] After these reactions, the DNA fragment 14 was inserted into thepW6A under the same conditions as in the step (e) in Example 1.

[0173] (c) The DNA fragment 4 prepared in the step (c) of Example 2 wassubjected to digestion at 37° C. for 1 hour with 5 units of theabove-mentioned Sal I and 5 units of the above-mentioned BamH.

[0174] The DNA fragment 14 inserted pW6A prepared in the step (b) ofExample 11 was also subjected to digestion at 37° C. for 1 hour with 5units of the above-mentioned Sal I and 5 units of the above-mentionedBamH I.

[0175] After these reactions, the DNA fraction 4 was inserted into theDNA fragment 14 inserted pW6A under the same conditions as in the step(e) in Example 1, whereby an expression vector for a 15-17-47 fusedantigen was prepared.

[0176] The results are shown in FIG. 5.

EXAMPLE 12 Preparation of Expression Vector for 47-17-15 Fused Antigen

[0177] (a) As a sense primer, Primer 1 prepared in Reference Example 3was selected, and as an antisense primer, Primer 18 prepared in Example3 was selected.

[0178] A mixture of each of the above-mentioned Primers 1 and 18 at afinal concentration of 1 μM, 0.1 ng of the 47K gene prepared inReference Example 4, and 2.5 units of the above-mentioned Taq polymerasewas subjected to the PCR under a heat application cycle of 94° C. for 1minute, 55° C. for 2 minutes and 72° C. for 3 minutes, with this heatapplication cycle being repeated 30 times, whereby a DNA fragment 15 wasprepared.

[0179] (b) The DNA fragment 15 prepared in the step (a) of Example 12was subjected to digestion at 37° C. for 1 hour with 5 units of theabove-mentioned Sac I (made by Toyobo Co., Ltd.) and then subjected todigestion at 37° C. for 1 hour with 5 units of the above-mentioned NdeI.

[0180] The pW6A prepared in Reference Example 2 was also subjected todigestion at 37° C. for 1 hour with 5 units of the above-mentioned Sac Iand then subjected to digestion at 37° C. for 1 hour with 5 units of theabove-mentioned Nde I.

[0181] After these reactions, the DNA fragment 15 was inserted into thepW6A under the same conditions as in the step (e) in Example 1.

[0182] (c) As a sense primer, Primer 19 prepared in Reference Example 3was selected, and as an antisense primer, Primer 28 prepared in Example3 was selected.

[0183] A mixture of each of the above-mentioned Primers 19 and 28 at afinal concentration of 1 μM, 0.1 ng of the 17K gene prepared inReference Example 4, and 2.5 units of the above-mentioned Taq polymerasewas subjected to PCR under a heat application cycle of 94° C. for 1minute, 55° C. for 2 minutes and 72° C. for 3 minutes, with this heatapplication cycle being repeated 30 times, whereby a DNA fragment 16 wasprepared.

[0184] (d) As a sense primer, Primer 29 prepared in Reference Example 3was selected, and as an antisense primer, Primer 9 prepared in Example 3was selected.

[0185] A mixture of each of the above-mentioned Primers 29 and 9 at afinal concentration of 1 μM, 0.1 ng of the 15K gene prepared inReference Example 4, and 2.5 units of the above-mentioned Taq polymerasewas subjected to PCR under a heat application cycle of 94° C. for 1minute, 55° C. for 2 minutes and 72° C. for 3 minutes, with this heatapplication cycle being repeated 30 times, whereby a DNA fragment 17 wasprepared.

[0186] (e) The DNA fragment 16 prepared in the step (c) of Example 12was subjected to digestion at 37° C. for 1 hour with 5 units of theabove-mentioned Sac I and then subjected to digestion at 37° C. for 1hour with 5 units of EcoR I (made by Toyobo Co., Ltd.).

[0187] The DNA fragment 17 prepared in the step (d) of Example 12 wassubjected to digestion at 37° C. for 1 hour with 5 units of theabove-mentioned EcoR I and 5 units of the above-mentioned BamH I.

[0188] The DNA fragment 15 inserted pW6A prepared in the step (b) ofExample 12 was subjected to digestion at 37° C. for 1 hour with 5 unitsof the above-mentioned Sac I and then subjected to digestion at 37° C.for 1 hour with 5 units of the above-mentioned BamH I.

[0189] After these reactions, the DNA fragment 16 and the DNA fragment17 were simultaneously inserted into the DNA fragment 15 inserted pW6Aunder the same conditions as in the step (e) in Example 1, whereby anexpression vector for a 47-17-15 fused antigen was obtained. The resultsare shown in FIG. 5.

EXAMPLE 13 Preparation of Expression Vector for 15 Fused Antigen

[0190] (a) As a sense primer, Primer 7 prepared in Reference Example 3was selected, and as an antisense primer, Primer 9 prepared in Example 3was selected.

[0191] A mixture of each of the above-mentioned Primers 7 and 9 at afinal concentration of 1 μM, 0.1 ng of the 15K gene prepared inReference Example 4, and 2.5 units of the above-mentioned Taq polymerasewas subjected to the PCR under a heat application cycle of 94° C. for 1minute, 55° C. for 2 minutes and 72° C. for 3 minutes, with this heatapplication cycle being repeated 30 times, whereby a DNA fragment 18 wasprepared.

[0192] (b) The DNA fragment 18 prepared in the step (a) of Example 13was subjected to digestion at 37° C. for 1 hour with 5 units of theabove-mentioned Nde I and 5 units of the above-mentioned BamH I.

[0193] The pW6A prepared in Reference Example 2 was subjected todigestion at 37° C. for 1 hour with 5 units of the above-mentioned Nde Iand 5 units of the above-mentioned BamH I.

[0194] After these reactions, the DNA fragment 18 was inserted into thepW6A under the same conditions as in the step (e) in Example 1, wherebyan expression vector for a 15 fused antigen was obtained. The resultsare shown in FIG. 6.

EXAMPLE 14 Preparation of Expression Vector for 15-15 Fused Antigen

[0195] (a) As a sense primer, Primer 11 prepared in Reference Example 3was selected, and as an antisense primer, Primer 9 prepared in Example 3was selected.

[0196] A mixture of each of the above-mentioned Primers 11 and 9 at afinal concentration of 1 μM, 0.1 ng of the 15K gene prepared inReference Example 4, and 2.5 units of the above-mentioned Taq polymerasewere was subjected to the PCR under a heat application cycle of 94° C.for 1 minute, 55° C. for 2 minutes and 72° C. for 3 minutes, with thisheat application cycle being repeated 30 times, whereby a DNA fragment19 was prepared.

[0197] (b) The DNA fragment 19 prepared in the step (a) of Example 14was subjected to digestion at 37° C. for 1 hour with 5 units of theabove-mentioned Sal I and 5 units of the above-mentioned BamH I.

[0198] The expression vector for the 15-47-17 fused antigen prepared inExample 10 was subjected to digestion at 37° C. for 1 hour with 5 unitsof the above-mentioned Sal I and 5 units of the above-mentioned BamH I.

[0199] After these reactions, the DNA fragment 19 was inserted into anexpression vector for a 15-47-17 fused antigen from which a DNA fragment47-17 was eliminated, under the same conditions as in the step (e) inExample 1, whereby an expression vector for a 15-15 fused antigen wasobtained. The results are shown in FIG. 6.

EXAMPLE 15 Preparation of Expression Vector for 15-15-15 Fused Antigen

[0200] (a) As a sense primer, Primer 11 prepared in Reference Example 3was selected, and as an antisense primer, Primer 22 prepared in Example3 was selected.

[0201] A mixture of each of the above-mentioned Primers 11 and 22 at afinal concentration of 1 μM, 0.1 ng of the 15K gene prepared inReference Example 4, and 2.5 units of the above-mentioned Taq polymerase(made by TaKaRa Co., Ltd.) was subjected to PCR under a heat applicationcycle of 94° C. for 1 minute, 55° C. for 2 minutes and 72° C. for 3minutes, with this heat application cycle being repeated 30 times,whereby a DNA fragment 20 was prepared.

[0202] (b) The DNA fragment 20 prepared in the step (a) of Example 15was subjected to digestion at 37° C. for 1 hour with 5 units of theabove-mentioned Sal I and 5 units of the above-mentioned BamH I.

[0203] The expression vector for the 15-47-17 fused antigen prepared inExample 10 was also subjected to digestion at 37° C. for 1 hour with 5units of the above-mentioned Sal I and 5 units of the above-mentionedBamH I.

[0204] After these reactions, the DNA fragment 20 was inserted into anexpression vector for a 15-47-17 fused antigen from which a DNA fragment47-17 was eliminated, under the same conditions as in the step (e) inExample 1, whereby a DNA fragment 15-15 inserted pW6A was prepared.

[0205] (c) As a sense primer, Primer 23 prepared in Reference Example 3was selected, and as an antisense primer, Primer 24 prepared in Example3 was selected.

[0206] A mixture of each of the above-mentioned Primers 23 and 24 at afinal concentration of 1 μM, 0.1 ng of the 15K gene prepared inReference Example 4, and 2.5 units of the above-mentioned Taq polymerase(made by TaKaRa Co., Ltd.) was subjected to PCR under a heat applicationcycle of 94° C. for 1 minute, 55° C. for 2 minutes and 72° C. for 3minutes, with this heat application cycle being repeated 30 times,whereby a DNA fragment 21 was prepared.

[0207] (d) The DNA fragment 21 prepared in step (c) of Example 15 wassubjected to digestion at 37° C. for 1 hour with 5 units of theabove-mentioned Kpn I (made by Toyobo Co., Ltd.) and then subjected todigestion at 37° C. for 1 hour with 5 units of the above-mentioned BamHI.

[0208] The DNA fragment 15-15 inserted pW6A prepared in step (b) ofExample 15 was also subjected to digestion at 37° C. for 1 hour with 5units of the above-mentioned Kpn I (made by Toyobo Co., Ltd.) and thensubjected to digestion at 37° C. for 1 hour with 5 units of theabove-mentioned BamH I.

[0209] After these reactions, the DNA fragment 21 was inserted into theDNA fragment 15-15 inserted pW6A under the same conditions as in step(e) in Example 1, whereby an expression vector for a 15-15-15 fusedantigen was prepared.

[0210] The results are shown in FIG. 6.

EXAMPLE 16 Preparation of Expression Vector for 15-15-15-15 FusedAntigen

[0211] (a) As a sense primer, Primer 23 prepared in Reference Example 3was selected, and as an antisense primer, Primer 25 prepared in Example3 was selected.

[0212] A mixture of each of the above-mentioned Primers 23 and 25 at afinal concentration of 1 μM, 0.1 ng of the 15K gene prepared inReference Example 4, and 2.5 units of the above-mentioned Taq polymerase(made by TaKaRa Co., Ltd.) was subjected to PCR under a heat applicationcycle of 94° C. for 1 minute, 55° C. for 2 minutes and 72° C. for 3minutes, with this heat application cycle being repeated 30 times,whereby a DNA fragment 25 was prepared.

[0213] (b) The DNA fragment 22 prepared in the step (a) of Example 16was subjected to digestion at 37° C. for 1 hour with 5 units of theabove-mentioned Kpn I and then subjected to digestion at 37° C. for 1hour with 5 units of the above-mentioned BamH I.

[0214] The DNA fragment 15-15 inserted in pW6A prepared in the step (b)of Example 15 was also subjected to digestion at 37° C. for 1 hour with5 units of the above-mentioned Kpn I and then subjected to digestion at37° C. for 1 hour with 5 units of the above-mentioned BamH I.

[0215] After these reactions, the DNA fragment 22 was inserted into theDNA fragment 15-15 inserted pW6A under the same conditions as in thestep (e) in Example 1, whereby a DNA fragment 15-15-15 inserted pW6A wasprepared. (c) As a sense primer, Primer 26 prepared in Reference Example3 was selected, and as an antisense primer, Primer 27 prepared inExample 3 was selected.

[0216] A mixture of each of the above-mentioned Primers 26 and 27 at afinal concentration of 1 μM, 0.1 ng of the 15K gene prepared inReference Example 4, and 2.5 units of the above-mentioned Taq polymerase(made by TaKaRa Co., Ltd.) was subjected to PCR under a heat applicationcycle of 94° C. for 1 minute, 55° C. for 2 minutes and 72° C. for 3minutes, with this heat application cycle being repeated 30 times,whereby a DNA fragment 23 was prepared.

[0217] (d) The DNA fragment 23 prepared in the step (c) of Example 16was subjected to digestion at 37° C. for 1 hour with 5 units of theabove-mentioned Kpn I and then subjected to digestion at 37° C. for 1hour with 5 units of the above-mentioned Hind III (made by Toyobo Co.Ltd.).

[0218] The DNA fragment 15-15-15 inserted pW6A prepared in the step (b)of Example 16 was also subjected to digestion at 37° C. for 1 hour with5 units of the above-mentioned Kpn I and then subjected to digestion at37° C. for 1 hour with 5 units of the above-mentioned Hind III (made byToyobo Co. Ltd.).

[0219] After these reactions, the DNA fragment 23 was inserted into theDNA fragment 15-15-15 inserted pW6A under the same conditions as in thestep (e) in Example 1, whereby an expression vector for a 15-15-15-15fused antigen was prepared.

[0220] The results are shown in FIG. 6.

EXAMPLE 17 Expression of Each Fused Antigen

[0221]Escherichia coli BL21 (DE3), which is a competent cell prepared byHanahan method, was transformed using each of the above-mentionedexpression vectors obtained in Examples 1 to 12.

[0222] The transformation culture thereof was then plated on an LB agarplate containing 50 μg/ml of ampicillin and incubated at 37° C.overnight. From the mixture of ampicillin-resistant Escherichia colicells thus produced, there was selected the transformant carrying anexpression vector for each fused antigen in which each fused antigengene was integrated.

[0223] Each of the thus selected transformant was then inoculated to 4ml of an LB liquid medium containing 50 μg/ml of ampicillin, andcultured with shaking at 37° C. overnight.

[0224] The thus obtained culture was further inoculated into 1 l of anLB liquid culture medium containing 50 μg/ml of ampicillin, and culturedwith shaking at 37° C. for 3 hours.

[0225] To the thus cultured Escherichia coli, IPTG(isopropyl-β-D(−)-thiogalactopyranoside) was added at a finalconcentration of 1 mM.

[0226] Cultivation was then further continued with shaking at 37° C.overnight, whereby the expression of each fused antigen was carried out.

EXAMPLE 18 Purification of Fused Antigens

[0227] 47-17-15 fused antigen, 47-15-17 fused antigen, 15-17-47 fusedantigen, and 15-17 fused antigen were purified.

[0228] The fused antigens of a three-antigen-fused type, namely,47-17-15 fused antigen, 47-15-17 fused antigen, and 15-17-47 fusedantigen were purified by the steps of subjecting each antigen-expressedEscherichia coli to ultrasonic blending and centrifugation, followed bymaking the residue soluble, using urea, and subjecting the antigen togel filtration chromatography using Superdex-200 (Pharmacia Biotec Co.,Ltd.), ion-exchange chromatography using Q-Sepharose (Pharmacia BiotecCo., Ltd.), and absorption chromatography using ceramic hydroxyapatite(made by Asahi Optical Co., Ltd.).

[0229] The fused antigen of a two-antigen-fused type, namely, 15-17fused antigen was purified by the steps of subjecting theantigen-expressed Escherichia coli to ultrasonic blending andcentrifugation, pooling the supernatant, adding urea thereto, andsubjecting the antigen to ion-exchange chromatography using Q-Sepharose(made by Pharmacia Biotec Co., Ltd.), absorption chromatography usingceramic hydroxyapatite (made by Asahi Optical Co., Ltd.), and thenion-exchange chromatography using SP-Sepharose (made by Pharmacia BiotecCo., Ltd.).

[0230] 7 mg of 47-17-15 fused antigen, 10 mg of 47-15-17 fused antigen,20 mg of 15-17-47 fused antigen and 10 mg of 15-17 fused antigen wereobtained with a purity of 95% or more from one litter of the respectiveculture media.

[0231] After the purification, each fused antigen was subjected toreduction SDS-polyacrylamide gel electrophoresis and then to coomassiestaining. The results are shown in FIG. 7.

[0232] It was expected that the antigens of the three-antigen-fused typehad a molecular weight of 75K and that 15-17 fused antigen had amolecular weight of 29K in view of the respective amino acid sequencesthereof. It was confirmed that the above expectation was correct by theelectrophoretic analyses thereof.

[0233] By Western blotting which was conducted by use of 47KMab, 17KMaband l5KMab which were prepared in Reference Example 1, it was confirmedthat these monoclonal antibodies were reactive with the correspondingantigens in each of the fused antigens.

EXAMPLE 19 Preparation of Fused Antigen Bound Plate

[0234] Each of the fused antigens purified in Example 18 was dilutedwith 10 mM PBS, placed in an amount of 0.66 pmol/well in each well of a96-well ELISA plate (made by Becton Dickinson Co., Ltd.) and allowed tostand at 4° C. overnight, whereby the fused antigen was bound to eachwell. After this binding, each fused antigen bound well was blocked with10 mM PBS containing 1% skim milk at 37° C. for 2 hours.

Reference Example 5 Preparation of Single Antigen Bound Plate

[0235] Each of a recombinant single antigen 47K antigen expressed byEscherichia coli and purified, a 17K antigen including a signal peptideat the N-terminus thereof (hereinafter referred to as S17K antigen), andGST15K antigen was bound in an amount of 0.66 pmol/wel to a well.

[0236] Furthermore, a mixture of the above-mentioned recombinant singleantigen 47K antigen, S17K antigen, and GST15K antigen was bound in anamount of 0.66 pmol/wel to a well.

[0237] After the above-mentioned binding, the wells were blocked with 10mM PBS containing 1% skim milk at 37° C. for 2 hours.

EXAMPLE 20 Tests for Reactivity between Fused Antigens and MonoclonalAntibodies

[0238] Each of 47KMab, 17KMab, 15KMab and a mixture thereof was dilutedwith 10 mM PBS containing 1% of skim milk and 0.05% Tween 20 (Trademark)(hereinafter referred to as the skim milk containing PBST) to such adilution degree that the final concentration of each monoclonal antigenwas 10 μm/ml.

[0239] Each of the above diluted monoclonal antigen and theabove-mentioned mixture was added in an amount of 50 μl to each of thewashed wells of the antigen-bound plates prepared in Example 19 andReference Example 5, and was then incubated at room temperature for 1hour and 30 minutes.

[0240] After each of the wells was washed, 50 μl of a peroxidase labeledanti-mouse Ig (made by DAKO Co., Ltd.) diluted to {fraction(1/1000)}with the skim milk containing PBST was added to each well, andthe mixture was incubated at room temperature for 1 hour and 30 minutes.

[0241] After each of the wells was washed, 50 μl of a mixed solution ofa hydrogen peroxide solution and ABTS was added thereto, and ABTS wascolored for 3 minutes. After the termination of the reaction, theabsorbance thereof with 405 nm thereof was measured by aspectrophotometer.

[0242] The results are shown in TABLE 1. As shown in TABLE 1, the fusedantigens exhibit the same activity as or higher activity than that ofany of the single antigens, 47K, S17K and GST15K. TABLE 1 47KMab +17KMab + 47KMab 17KMab 15KMab 15KMab Fused 47-17-15 0.56 0.35 0.47 1.48Anti- 47-15-17 1.26 0.51 0.52 1.53 gens 15-17-47 1.21 0.76 1.26 1.8915-17 — 0.13 0.60 0.36 Single 47K 0.52 — — — Anti- S17K — 0.05 — — gensGST15K — — 0.45 — 47K + S17K + GST15K — — — 0.95

EXAMPLE 21 Comparative Tests Concerning Reactivities between FusedAntigens and Single Antigens

[0243] 50 μl of each of a Tp positive serum (purchased from BostonBiomedica Inc.) which was diluted to {fraction (1/1000)}with the skimmilk containing PBST, and a Tp negative serum was added to each well ofthe washed antigen bound plates which were prepared in Example 19 andReference Example 5

[0244] Each of the above-mentioned serums was incubated at roomtemperature for 1 hour and 30 minutes.

[0245] After each of the wells was washed, 50 μl of a peroxidase labeledantihuman Ig G (made by BIO SOURCE Co., Ltd.) diluted to {fraction(1/1000)}with the skim milk containing PBST was added to each well, andthe mixture was incubated at room temperature for 1 hour and 30 minutes.

[0246] After each of the wells was washed, 50 μl of a mixed solution ofa hydrogen peroxide solution and ABTS was added thereto, and ABTS wascolored for 3 minutes. After the termination of the reaction, theabsorbance thereof with 405 nm thereof was measured by aspectrophotometer.

[0247] The results are shown in TABLE 2. As shown in TABLE 2, the fusedantigens exhibit higher reactivity with the positive serum than thesingle antigens and the mixture thereof, and never react with thenegative serum. TABLE 2 Positive Serum Negative Serum Fused 47-17-150.56 0.02 Anti- 47-15-17 1.18 0.02 gens 15-17-47 1.13 0.03 15-17 0.440.01 Single 47K 0.13 0.03 Anti- S17K 0.11 0.01 gens GST15K 0.16 0.0247K + S17K + GST15K 0.39 0.03

EXAMPLE 22 Reactivity Tests between Fused Antigens and Positive Serum

[0248] 24 examples of Tp positive serums (purchased from BostonBiomedica Inc.) and one example of Tp negative serum were tested in thesame manner as in Example 21 except that the coloring time was extendedto 30 minutes.

[0249] The results are shown in TABLE 3. The reactivities of the fusedantigens with 24 examples of the positive serums were significantlyhigher than that of the fused antigens with the negative serum. TABLE 3Solid Phase Antigens 47-17-15 47-15-17 15-17-47 15-17 Positive No. 10.54 1.15 1.85 0.81 Serums No. 2 >2 >2 >2 1.47 No. 3 1.51 >2 >2 1.72 No.4 >2 >2 >2 >2 No. 5 0.52 1.29 1.57 0.95 No. 6 0.34 0.64 1.08 0.35 No.7 >2 >2 >2 >2 No. 8 >2 >2 >2 >2 No. 9 0.56 1.05 1.40 0.69 No.10 >2 >2 >2 >2 No. 11 >2 >2 >2 >2 No. 12 1.30 >2 >2 1.21 No. 131.79 >2 >2 1.55 No. 14 >2 >2 >2 >2 No. 15 >2 >2 >2 >2 No. 16 >2 >2 >2 >2No. 17 1.39 >2 >2 1.25 No. 18 >2 >2 >2 >2 No. 19 1.62 >2 >2 1.29 No. 200.64 1.08 1.28 0.73 No. 21 >2 >2 >2 >2 No. 22 0.42 1.11 1.41 0.77 No. 230.57 1.52 1.43 0.91 No. 24 >2 >2 >2 >2 Negative Serum 0.06 0.08 0.070.06

EXAMPLE 23 Expression of 15K, 15K-15K, 15K-15K-15K and 15K-15K-15K-15K

[0250]Escherichia coli BL21 (DE3) was transformed using each of the 15K,15K-15K, 15K-15K-15K and 15K-15K-15K-15K expression vectors obtained inExamples 13 to 16.

[0251] The transformation culture thereof was then plated on an LB agarplate containing 50 μg/ml of ampicillin and incubated at 37° C.overnight. From the mixture of ampicillin-resistant Escherichia colicells thus produced, the transformant was selected carrying anexpression vector for each fused antigen in which each fused antigengene was integrated.

[0252] Each of the thus selected transformants was inoculated to 2 ml ofan LB liquid medium containing 50 μg/ml of ampicillin, and cultured withshaking at 37° C. overnight.

[0253] 40 μl of the thus obtained culture was inoculated to 2 ml of anLB liquid medium containing 50 μg/ml of ampicillin and further culturedwith shaking at 37° C. for 2 hours. To the thus cultured Escherichiacoli, IPTG (isopropyl-β-D(−)-thiogalactopyranoside) was added at a finalconcentration of 1 mM.

[0254] Cultivation was then further continued with shaking at 37° C.overnight, whereby the expression of each fused antigen was carried out.

[0255] Out of 2 ml of the thus obtained culture, 1.5 ml thereof wastransferred into a micro centrifugation tube and centrifuged at 15000rpm for 30 seconds by use of a micro centrifuge (Trademark “himac CT15D” made by Hitachi Ltd.), whereby a cell pellet of the desiredEscherichia coli transformant was collected.

[0256] Each cell pellet was resuspended in 150 μl of TE Buffer (10 mMtris-hydrochloric acid buffer, pH8.0, 0.1 mM, EDTA), and 150 μl of x2sample buffer (1M tris-hydrochloric acid buffer, pH6.8, 20%w/v SDS,10%v/v β-ME), and boiled for 5 minutes. Then, each sample was subjectedto ultrasonic treatment for 10 minutes.

[0257] To 13 μl of the thus obtained sample, 2 μl of 50% glycerin wasadded, and the mixture was mixed. 10 μl of the mixture was thenelectrophoresed on the 15% SDS-polyacrylamide gel under the reducingcondition (Laemmli, U.K. 1970: Nature 227, 680), and was coomassie blueR stained.

[0258] The results are shown in FIG. 8.

[0259] By the overnight expression, the presence of 15K-15K, 15K-15K-15Kand 15K-15K-15K-15K bands was confirmed.

[0260] Furthermore, each antigen expressed overnight was also subjectedto SDS-PAGE in the same manner as mentioned above on the 10 to 15%polyacrylic amide gradient gel, and then subjected to western blottingwith the 15KMab prepared in Reference Example 1.

[0261] The result was that as shown in FIG. 9, the presence of all ofthe 15K, 15K-15K, 15K-15K-15K and 15K-15K-15K-15K bands was confirmed.

EXAMPLE 24 Comparison among Expression Amounts of 15K, 15K-15K,15K-15K-15K and 15K-15K-15K-15K

[0262] An electrophoresis sample which was prepared as shown in Example23, and molecular mass standards containing a known concentration of BSA(Trademark “Low Molecular Weight Calibration Kit” made by Pharmacia LKB)were electrophoresed on the 15% SDS-polyacrylamide gel under reducingconditions, and were coomassie blue R stained. The results are shown inFIG. 10.

[0263] The amounts of the expressed antigens were calculated from thestained bands thereof by use of a densitometer (Trademark “DensitographAE-6900” made by Atto Co., Ltd.)

[0264] From the coomassie blue R stained gel in FIG. 10, the density ofthe bands of BSA with a known concentration was measured by thedensitometer, whereby a working curve as shown in FIG. 11 was obtained.Furthermore, the densities of the bands corresponding to the 15K,15K-15K, 15K-15K-15K and 15K-15K-15K-15K were also measured in the samemanner as mentioned above by use of the densitometer, and the respectiveexpression amounts were calculated by use of the working curve shown inFIG. 11. The results are shown in TABLE 4 as shown below. TABLE 4 PeakArea Concentration of Kinds of Antigens (Arbitrary Unit) Protein(μg/lane) 15K n.d n.d 15K-15K 499 0.34 15K-15K-15K 856 1.0 15K-15K-15K-15K 436 0.27

[0265] With respect to the 15K, no bands were observed. In contrast tothis, with respect to the 15K-15K-15K, the expression amount thereof was1 μg per lane (corresponding to 23 mg per 1 litter of the culture),which was the largest of all the tested samples. By fusing 15Krepeatedly in this manner, the expression amount thereof wassignificantly increased.

[0266] Japanese Patent Application No. 7-350072 filed Dec. 25, 1995 ishereby incorporated by reference.

What is claimed is:
 1. A Treponema pallidum fused antigen in which atleast two surface antigens of Treponema pallidum are fused.
 2. ATreponema pallidum fused antigen in which at least three surfaceantigens of Treponema pallidum are fused.
 3. A Treponema pallidum fusedantigen in which two to three surface antigens of Treponema pallidum arefused.
 4. The Treponema pallidum fused antigen as claimed in claim 1 ,wherein said at least two surface antigens of Treponema pallidum have amolecular weight selected from 47kDa, 17kDa and 15kDa.
 5. The Treponemapallidum fused antigen as claimed in claim 2 , wherein said at leastthree surface antigens of Treponema pallidum have a molecular weightselected from 47kDa, 17kDa and 15kDa.
 6. The Treponema pallidum fusedantigen as claimed in claim 3 , wherein said two to three surfaceantigens of Treponema pallidum have a molecular weight selected from47kDa, 17kDa and 15kDa.
 7. The Treponema pallidum fused antigen asclaimed in claim 4 , wherein said surface antigens are fused in theorder of said surface antigen with a molecular weight of 47kDa, saidsurface antigen with a molecular weight of 17kDa, and said surfaceantigen with a molecular weight of 15kDa in view of the sequence from anN-terminus thereof to a C-terminus thereof; in the order of said surfaceantigen with a molecular weight of 47kDa, said surface antigen with amolecular weight of 15kDa, and said surface antigen with a molecularweight of 17kDa in view of the sequence from an N-terminus thereof to aC-terminus thereof; in the order of said surface antigen with amolecular weight of 15kDa, said surface antigen with a molecular weightof 17kDa, and said surface antigen with a molecular weight of 47kDa inview of the sequence from an N-terminus thereof to a C-terminus thereof;in the order of said surface antigen with a molecular weight of 15kDa,said surface antigen with a molecular weight of 47kDa, and said surfaceantigen with a molecular weight of 17kDa in view of the sequence from anN-terminus thereof to a C-terminus thereof; in the order of said surfaceantigen with a molecular weight of 17kDa, said surface antigen with amolecular weight of 15kDa, and said surface antigen with a molecularweight of 47kDa in view of the sequence from an N-terminus thereof to aC-terminus thereof; in the order of said surface antigen with amolecular weight of 17kDa, said surface antigen with a molecular weightof 47kDa, and said surface antigen with a molecular weight of 15kDa inview of the sequence from an N-terminus thereof to a C-terminus thereof;in the order of said surface antigen with a molecular weight of 47kDaand said surface antigen with a molecular weight of 17kDa in view of thesequence from an N-terminus thereof to a C-terminus thereof; in theorder of said surface antigen with a molecular weight of 47kDa and saidsurface antigen with a molecular weight of 15kDa in view of the sequencefrom an N-terminus thereof to a C-terminus thereof; in the order of saidsurface antigen with a molecular weight of 17kDa and said surfaceantigen with a molecular weight of 47kDa in view of the sequence from anN-terminus thereof to a C-terminus thereof; in the order of said surfaceantigen with a molecular weight of 17kDa and said surface antigen with amolecular weight of 15kDa in view of the sequence from an N-terminusthereof to a C-terminus thereof; in the order of said surface antigenwith a molecular weight of 15kDa and said surface antigen with amolecular weight of 47kDa in view of the sequence from an N-terminusthereof to a C-terminus thereof; in the order of said surface antigenwith a molecular weight of 15kDa and said surface antigen with amolecular weight of 17kDa in view of the sequence from an N-terminusthereof to a C-terminus thereof; in the order of said surface antigenwith a molecular weight of 15kDa and said surface antigen with amolecular weight of 15kDa in view of the sequence from an N-terminusthereof to a C-terminus thereof; in the order of said surface antigenwith a molecular weight of 15kDa, said surface antigen with a molecularweight of 15kDa and said surface antigen with a molecular weight of15kDa in view of the sequence from an N-terminus thereof to a C-terminusthereof; or in the order of said surface antigen with a molecular weightof 15kDa, said surface antigen with a molecular weight of 15kDa, saidsurface antigen with a molecular weight of 15kDa and said surfaceantigen with a molecular weight of 15kDa in view of the sequence from anN-terminus thereof to a C-terminus thereof.
 8. The Treponema pallidumfused antigen as claimed in claim 5 , wherein said surface antigens arefused in the order of said surface antigen with a molecular weight of47kDa, said surface antigen with a molecular weight of 17kDa, and saidsurface antigen with a molecular weight of 15kDa in view of the sequencefrom an N-terminus thereof to a C-terminus thereof; in the order of saidsurface antigen with a molecular weight of 47kDa, said surface antigenwith a molecular weight of 15kDa, and said surface antigen with amolecular weight of 17kDa in view of the sequence from an N-terminusthereof to a C-terminus thereof; in the order of said surface antigenwith a molecular weight of 15kDa, said surface antigen with a molecularweight of 17kDa, and said surface antigen with a molecular weight of47kDa in view of the sequence from an N-terminus thereof to a C-terminusthereof; in the order of said surface antigen with a molecular weight of15kDa, said surface antigen with a molecular weight of 47kDa, and saidsurface antigen with a molecular weight of 17kDa in view of the sequencefrom an N-terminus thereof to a C-terminus thereof; in the order of saidsurface antigen with a molecular weight of 17kDa, said surface antigenwith a molecular weight of 15kDa, and said surface antigen with amolecular weight of 47kDa in view of the sequence from an N-terminusthereof to a C-terminus thereof; in the order of said surface antigenwith a molecular weight of 17kDa, said surface antigen with a molecularweight of 47kDa, and said surface antigen with a molecular weight of15kDa in view of the sequence from an N-terminus thereof to a C-terminusthereof; in the order of said surface antigen with a molecular weight of47kDa and said surface antigen with a molecular weight of 17kDa in viewof the sequence from an N-terminus thereof to a C-terminus thereof; inthe order of said surface antigen with a molecular weight of 47kDa andsaid surface antigen with a molecular weight of 15kDa in view of thesequence from an N-terminus thereof to a C-terminus thereof; in theorder of said surface antigen with a molecular weight of 17kDa and saidsurface antigen with a molecular weight of 47kDa in view of the sequencefrom an N-terminus thereof to a C-terminus thereof; in the order of saidsurface antigen with a molecular weight of 17kDa and said surfaceantigen with a molecular weight of 15kDa in view of the sequence from anN-terminus thereof to a C-terminus thereof; in the order of said surfaceantigen with a molecular weight of 15kDa and said surface antigen with amolecular weight of 47kDa in view of the sequence from an N-terminusthereof to a C-terminus thereof; in the order of said surface antigenwith a molecular weight of 15kDa and said surface antigen with amolecular weight of 17kDa in view of the sequence from an N-terminusthereof to a C-terminus thereof; in the order of said surface antigenwith a molecular weight of 15kDa and said surface antigen with amolecular weight of 15kDa in view of the sequence from an N-terminusthereof to a C-terminus thereof; in the order of said surface antigenwith a molecular weight of 15kDa, said surface antigen with a molecularweight of 15kDa and said surface antigen with a molecular weight of15kDa in view of the sequence from an N-terminus thereof to a C-terminusthereof; or in the order of said surface antigen with a molecular weightof 15kDa, said surface antigen with a molecular weight of 15kDa, saidsurface antigen with a molecular weight of 15kDa and said surfaceantigen with a molecular weight of 15kDa in view of the sequence from anN-terminus thereof to a C-terminus thereof.
 9. The Treponema pallidumfused antigen as claimed in claim 6 , wherein said surface antigens arefused in the order of said surface antigen with a molecular weight of47kDa, said surface antigen with a molecular weight of 17kDa, and saidsurface antigen with a molecular weight of 15kDa in view of the sequencefrom an N-terminus thereof to a C-terminus thereof; in the order of saidsurface antigen with a molecular weight of 47kDa, said surface antigenwith a molecular weight of 15kDa, and said surface antigen with amolecular weight of 17kDa in view of the sequence from an N-terminusthereof to a C-terminus thereof; in the order of said surface antigenwith a molecular weight of 15kDa, said surface antigen with a molecularweight of 17kDa, and said surface antigen with a molecular weight of47kDa in view of the sequence from an N-terminus thereof to a C-terminusthereof; in the order of said surface antigen with a molecular weight of15kDa, said surface antigen with a molecular weight of 47kDa, and saidsurface antigen with a molecular weight of 17kDa in view of the sequencefrom an N-terminus thereof to a C-terminus thereof; in the order of saidsurface antigen with a molecular weight of 17kDa, said surface antigenwith a molecular weight of 15kDa, and said surface antigen with amolecular weight of 47kDa in view of the sequence from an N-terminusthereof to a-C-terminus thereof; in the order of said surface antigenwith a molecular weight of 17kDa, said surface antigen with a molecularweight of 47kDa, and said surface antigen with a molecular weight of15kDa in view of the sequence from an N-terminus thereof to a C-terminusthereof; in the order of said surface antigen with a molecular weight of47kDa and said surface antigen with a molecular weight of 17kDa in viewof the sequence from an N-terminus thereof to a C-terminus thereof; inthe order of said surface antigen with a molecular weight of 47kDa andsaid surface antigen with a molecular weight of 15kDa in view of thesequence from an N-terminus thereof to a C-terminus thereof; in theorder of said surface antigen with a molecular weight of 17kDa and saidsurface antigen with a molecular weight of 47kDa in view of the sequencefrom an N-terminus thereof to a C-terminus thereof; in the order of saidsurface antigen with a molecular weight of 17kDa and said surfaceantigen with a molecular weight of 15kDa in view of the sequence from anN-terminus thereof to a C-terminus thereof; in the order of said surfaceantigen with a molecular weight of 15kDa and said surface antigen with amolecular weight of 47kDa in view of the sequence from an N-terminusthereof to a C-terminus thereof; in the order of said surface antigenwith a molecular weight of 15kDa and said surface antigen with amolecular weight of 17kDa in view of the sequence from an N-terminusthereof to a C-terminus thereof; in the order of said surface antigenwith a molecular weight of 15kDa and said surface antigen with amolecular weight of 15kDa in view of the sequence from an N-terminusthereof to a C-terminus thereof; in the order of said surface antigenwith a molecular weight of 15kDa, said surface antigen with a molecularweight of 15kDa and said surface antigen with a molecular weight of15kDa in view of the sequence from an N-terminus thereof to a C-terminusthereof; or in the order of said surface antigen with a molecular weightof 15kDa, said surface antigen with a molecular weight of 15kDa, saidsurface antigen with a molecular weight of 15kDa and said surfaceantigen with a molecular weight of 15kDa in view of the sequence from anN-terminus thereof to a C-terminus thereof.
 10. An assay foranti-Treponema pallidum antibodies, comprising reacting a Treponemapallidum fused antigen in which at least two surface antigens ofTreponema pallidum are fused with a sample in which said antibodies areto be detected.
 11. An assay for anti-Treponema pallidum antibodies,comprising reacting a Treponema pallidum fused antigen in which at leastthree surface antigens of Treponema pallidum are fused with a sample inwhich said antibodies are to be detected.
 12. An assay foranti-Treponema pallidum anti-bodies, comprising reacting a Treponemapallidum fused antigen in which two to three surface antigens ofTreponema pallidum are fused with a sample in which said antibodies areto be detected.
 13. The assay for anti-Treponema pallidum antibodies asclaimed in claim 10 , wherein said at least two surface antigens ofTreponema pallidum have a molecular weight selected from 47kDa, 17kDaand 15kDa.
 14. The assay for anti-Treponema pallidum antibodies asclaimed in claim 11 , wherein said at least three surface antigens ofTreponema pallidum have a molecular weight selected from 47kDa, 17kDaand 15kDa.
 15. The assay for anti-Treponema pallidum antibodies asclaimed in claim 12 , wherein said two to three surface antigens ofTreponema pallidum have a molecular weight selected from 47kDa, 17kDaand 15kDa.
 16. The assay for anti-Treponema pallidum antibodies asclaimed in claim 13 , wherein said surface antigens are fused in theorder of said surface antigen with a molecular weight of 47kDa, saidsurface antigen with a molecular weight of 17kDa, and said surfaceantigen with a molecular weight of 15kDa in view of the sequence from anN-terminus thereof to a C-terminus thereof; in the order of said surfaceantigen with a molecular weight of 47kDa, said surface antigen with amolecular weight of 15kDa, and said surface antigen with a molecularweight of 17kDa in view of the sequence from an N-terminus thereof to aC-terminus thereof; in the order of said surface antigen with amolecular weight of 15kDa, said surface antigen with a molecular weightof 17kDa, and said surface antigen with a molecular weight of 47kDa inview of the sequence from an N-terminus thereof to a C-terminus thereof;in the order of said surface antigen with a molecular weight of 15kDa,said surface antigen with a molecular weight of 47kDa, and said surfaceantigen with a molecular weight of 17kDa in view of the sequence from anN-terminus thereof to a C-terminus thereof; in the order of said surfaceantigen with a molecular weight of 17kDa, said surface antigen with amolecular weight of 15kDa, and said surface antigen with a molecularweight of 47kDa in view of the sequence from an N-terminus thereof to aC-terminus thereof; in the order of said surface antigen with amolecular weight of 17kDa, said surface antigen with a molecular weightof 47kDa, and said surface antigen with a molecular weight of 15kDa inview of the sequence from an N-terminus thereof to a C-terminus thereof;in the order of said surface antigen with a molecular weight of 47kDaand said surface antigen with a molecular weight of 17kDa in view of thesequence from an N-terminus thereof to a C-terminus thereof; in theorder of said surface antigen with a molecular weight of 47kDa and saidsurface antigen with a molecular weight of 15kDa in view of the sequencefrom an N-terminus thereof to a C-terminus thereof; in the order of saidsurface antigen with a molecular weight of 17kDa and said surfaceantigen with a molecular weight of 47kDa in view of the sequence from anN-terminus thereof to a C-terminus thereof; in the order of said surfaceantigen with a molecular weight of 17kDa and said surface antigen with amolecular weight of 15kDa in view of the sequence from an N-terminusthereof to a C-terminus thereof; in the order of said surface antigenwith a molecular weight of 15kDa and said surface antigen with amolecular weight of 47kDa in view of the sequence from an N-terminusthereof to a C-terminus thereof; in the order of said surface antigenwith a molecular weight of 15kDa and said surface antigen with amolecular weight of 17kDa in view of the sequence from an N-terminusthereof to a C-terminus thereof; in the order of said surface antigenwith a molecular weight of 15kDa and said surface antigen with amolecular weight of 15kDa in view of the sequence from an N-terminusthereof to a C-terminus thereof; in the order of said surface antigenwith a molecular weight of 15kDa, said surface antigen with a molecularweight of 15kDa and said surface antigen with a molecular weight of15kDa in view of the sequence from an N-terminus thereof to a C-terminusthereof; or in the order of said surface antigen with a molecular weightof 15kDa, said surface antigen with a molecular weight of 15kDa, saidsurface antigen with a molecular weight of 15kDa and said surfaceantigen with a molecular weight of 15kDa in view of the sequence from anN-terminus thereof to a C-terminus thereof.
 17. The assay foranti-Treponema pallidum antibodies as claimed in claim 14 , wherein saidsurface antigens are fused in the order of said surface antigen with amolecular weight of 47kDa, said surface antigen with a molecular weightof 17kDa, and said surface antigen with a molecular weight of 15kDa inview of the sequence from an N-terminus thereof to a C-terminus thereof;in the order of said surface antigen with a molecular weight of 47kDa,said surface antigen with a molecular weight of 15kDa, and said surfaceantigen with a molecular weight of 17kDa in view of the sequence from anN-terminus thereof to a C-terminus thereof; in the order of said surfaceantigen with a molecular weight of 15kDa, said surface antigen with amolecular weight of 17kDa, and said surface antigen with a molecularweight of 47kDa in view of the sequence from an N-terminus thereof to aC-terminus thereof; in the order of said surface antigen with amolecular weight of 15kDa, said surface antigen with a molecular weightof 47kDa, and said surface antigen with a molecular weight of 17kDa inview of the sequence from an N-terminus thereof to a C-terminus thereof;in the order of said surface antigen with a molecular weight of 17kDa,said surface antigen with a molecular weight of 15kDa, and said surfaceantigen with a molecular weight of 47kDa in view of the sequence from anN-terminus thereof to a C-terminus thereof; in the order of said surfaceantigen with a molecular weight of 17kDa, said surface antigen with amolecular weight of 47kDa, and said surface antigen with a molecularweight of 15kDa in view of the sequence from an N-terminus thereof to aC-terminus thereof; in the order of said surface antigen with amolecular weight of 47kDa and said surface antigen with a molecularweight of 17kDa in view of the sequence from an N-terminus thereof to aC-terminus thereof; in the order of said surface antigen with amolecular weight of 47kDa and said surface antigen with a molecularweight of 15kDa in view of the sequence from an N-terminus thereof to aC-terminus thereof; in the order of said surface antigen with amolecular weight of 17kDa and said surface antigen with a molecularweight of 47kDa in view of the sequence from an N-terminus thereof to aC-terminus thereof; in the order of said surface antigen with amolecular weight of 17kDa and said surface antigen with a molecularweight of 15kDa in view of the sequence from an N-terminus thereof to aC-terminus thereof; in the order of said surface antigen with amolecular weight of 15kDa and said surface antigen with a molecularweight of 47kDa in view of the sequence from an N-terminus thereof to aC-terminus thereof; in the order of said surface antigen with amolecular weight of 15kDa and said surface antigen with a molecularweight of 17kDa in view of the sequence from an N-terminus thereof to aC-terminus thereof; in the order of said surface antigen with amolecular weight of 15kDa and said surface antigen with a molecularweight of 15kDa in view of the sequence from an N-terminus thereof to aC-terminus thereof; in the order of said surface antigen with amolecular weight of 15kDa, said surface antigen with a molecular weightof 15kDa and said surface antigen with a molecular weight of 15kDa inview of the sequence from an N-terminus thereof to a C-terminus thereof;or in the order of said surface antigen with a molecular weight of15kDa, said surface antigen with a molecular weight of 15kDa, saidsurface antigen with a molecular weight of 15kDa and said surfaceantigen with a molecular weight of 15kDa in view of the sequence from anN-terminus thereof to a C-terminus thereof.
 18. The assay foranti-Treponema pallidum antibodies as claimed in claim 15 , wherein saidsurface antigens are fused in the order of said surface antigen with amolecular weight of 47kDa, said surface antigen with a molecular weightof 17kDa, and said surface antigen with a molecular weight of 15kDa inview of the sequence from an N-terminus thereof to a C-terminus thereof;in the order of said surface antigen with a molecular weight of 47kDa,said surface antigen with a molecular weight of 15kDa, and said surfaceantigen with a molecular weight of 17kDa in view of the sequence from anN-terminus thereof to a C-terminus thereof; in the order of said surfaceantigen with a molecular weight of 15kDa, said surface antigen with amolecular weight of 17kDa, and said surface antigen with a molecularweight of 47kDa in view of the sequence from an N-terminus thereof to aC-terminus thereof; in the order of said surface antigen with amolecular weight of 15kDa, said surface antigen with a molecular weightof 47kDa, and said surface antigen with a molecular weight of 17kDa inview of the sequence from an N-terminus thereof to a C-terminus thereof;in the order of said surface antigen with a molecular weight of 17kDa,said surface antigen with a molecular weight of 15kDa, and said surfaceantigen with a molecular weight of 47kDa in view of the sequence from anN-terminus thereof to a C-terminus thereof; in the order of said surfaceantigen with a molecular weight of 17kDa, said surface antigen with amolecular weight of 47kDa, and said surface antigen with a molecularweight of 15kDa in view of the sequence from an N-terminus thereof to aC-terminus thereof; in the order of said surface antigen with amolecular weight of 47kDa and said surface antigen with a molecularweight of 17kDa in view of the sequence from an N-terminus thereof to aC-terminus thereof; in the order of said surface antigen with amolecular weight of 47kDa and said surface antigen with a molecularweight of 15kDa in view of the sequence from an N-terminus thereof to aC-terminus thereof; in the order of said surface antigen with amolecular weight of 17kDa and said surface antigen with a molecularweight of 47kDa in view of the sequence from an N-terminus thereof to aC-terminus thereof; in the order of said surface antigen with amolecular weight of 17kDa and said surface antigen with a molecularweight of 15kDa in view of the sequence from an N-terminus thereof to aC-terminus thereof; in the order of said surface antigen with amolecular weight of 15kDa and said surface antigen with a molecularweight of 47kDa in view of the sequence from an N-terminus thereof to aC-terminus thereof; in the order of said surface antigen with amolecular weight of 15kDa and said surface antigen with a molecularweight of 17kDa in view of the sequence from an N-terminus thereof to aC-terminus thereof; in the order of said surface antigen with amolecular weight of 15kDa and said surface antigen with a molecularweight of 15kDa in view of the sequence from an N-terminus thereof to aC-terminus thereof; in the order of said surface antigen with amolecular weight of 15kDa, said surface antigen with a molecular weightof 15kDa and said surface antigen with a molecular weight of 15kDa inview of the sequence from an N-terminus thereof to a C-terminus thereof;or in the order of said surface antigen with a molecular weight of15kDa, said surface antigen with a molecular weight of 15kDa, saidsurface antigen with a molecular weight of 15kDa and said surfaceantigen with a molecular weight of 15kDa in view of the sequence from anN-terminus thereof to a C-terminus thereof.